Summary of dissertation Environmental engineering: Concentrative assessment of organic chlorine pesticides in water, sediments, aquatic organisms at the Saigon – Dong Nai River estuary and testing the toxicity of DDTs on embryos and larvae of Pacific oysters, Fish Medaka

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The objective of the dissertation is to determine the residue of pesticides OCPs in water, sediments, aquatic organisms at the estuary of the Saigon – Dong Nai and to evaluate the toxicity of the pesticides DDTs on the embryos and larvae of the Pacific oysters (Crassostrea Gigas), Fish Medaka (Oryzias latipes).

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Nội dung Text: Summary of dissertation Environmental engineering: Concentrative assessment of organic chlorine pesticides in water, sediments, aquatic organisms at the Saigon – Dong Nai River estuary and testing the toxicity of DDTs on embryos and larvae of Pacific oysters, Fish Medaka

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- Nguyen Xuan Tong ASSESSMENT OF ORGANIC CHLORINE PESTICIDES IN WATER, SEDIMENTS, AQUATIC ORGANISMS AT SAI GON-DONG NAI RIVER ESTUARY AND TESTING THE TOXICITY OF DDTs ON EMBRYOS AND LARVAE OF PACIFIC OYSTERS, FISH MEDAKA Major: Environmental Engineering Code: 9 52 03 20 SUMMARY OF DISSERTATION Hanoi – 2021
The dissertation was implemented at: Graduate University of Science and Technology - Vietnam Academy of Science and Technology. The first supervisor: Dr. Mai Huong The second supervisor: Assoc. Prof. Dr. Duong Thi Thuy Reviewer 1: … Reviewer 2: … Reviewer 3: …. The thesis will be defended at the Academy-level Ph.D. Thesis Evaluation Council, Graduate University of Science and Technology - Vietnam Academy of Science and Technology …. Stored by: - Library of Vietnam Academy of Science and Technology - National Library of Vietnam
1 INTRODUCTION 1. The necessary of the dissertation Organic component pesticides (OCPs) have been widely used around the world over the past centuries to control pests, fungi, and various insects to increase productivity, protect public health, and prevent mosquitoes that cause malaria. However, OCPs were banned or restricted from use globally several decades ago due to their toxicity to living organisms. Recently, rapidly growing industrial zones and urban areas can be potential sources of OCPs. The illegal uses of OCPs upstream have also caused the rise of OCPs concentrations in surface water and sediment in downstream areas of the Saigon – Dong Nai River. Thus, studies on OCPs in surface water, sediment, and estuarine species are really important. Therefore, the current thesis “Concentrative assessment of organic chlorine pesticides in water, sediments, aquatic organisms at the Saigon – Dong Nai River estuary and testing the toxicity of DDTs on embryos and larvae of Pacific oysters, Fish Medaka” was conducted from 2017 to 2018. 2. The objectives of the dissertation The objective of the dissertation is to determine the residue of pesticides OCPs in water, sediments, aquatic organisms at the estuary of the Saigon – Dong Nai and to evaluate the toxicity of the pesticides DDTs on the embryos and larvae of the Pacific oysters (Crassostrea Gigas), Fish Medaka (Oryzias latipes). 3. The main research contents of the dissertation To survey the current state of OCPs in water, sediments in the Saigon – Dong Nai estuary according to seasons and pollution groups. To investigate the current status of pesticide pollution of OCPs for fish, bivalve molluscs and to determine the source of pollution at the Saigon – Dong Nai river estuary. To evaluate the toxicity of the insecticide DDTs on the growth of Pacific oysters (Crassostrea Gigas) and Fish Medaka (Oryzias latipes) through the determination of LC50/EC50 and assessment of the effects on embryonic and larval morphology. CHAPTER 1: OVERVIEW 1.1. Overview on pesticides 1.2. Research situation and the state of pesticide residues in aquatic ecological environment 1.3. Overview on Pacific oysters (Crassostrea Gigas), Fish Medaka (Oryzias latipes) and their applications in ecological toxicity assessment 1.4. Overview on the study areas CHAPTER 2: RESEARCH METHODS 2.1. Chemicals, instruments, and laboratory equipment 2.2. Sampling location 2.3. Sampling methods 2.4. Sample analysis method 2.4.1. Analysis of physical and chemical parameters Table 2. 1. Technical analysis of physical and chemical parameters of surface water and sediment samples Matrix Physical and chemical parameters Technical analysis pH, Electrical Conductivity (EC), Total Hydrolab Model (Multi Set 430iWTW) Water Dissolved Solids (TDS), Temperature Turbidity Secchi plate (30 cm diameter)
2 Shaking 10 g of dry sediment in 25 mL of water for 10 minutes. Settling 10 minutes, pH measured with an electronic pH meter (HI 8424, Hanna Instruments, Sarmeola di Rubano PD, Italy) Sediment Total carbon analyzer (Multi C / N 3000, Total Organic Carbon (TOC) Analytik Jena AG, Jena, Germany) Microtrac S3500 laser particle size Particle size analyzer (Microtrac Inc., Montgomeryville, PA, USA) 2.4.2. Determination of OCPs in water samples 50 mL n–hexan was placed in 2 liters - separating funnel which containing 1 liter of distilled water and shaken manually for 5 minutes and then allowed to settle. After complete extraction, the organic phase is drained into a 250 ml conical flask, while the aqueous phase was extracted twice with 50 ml n-hexane. The three extracted organic phases were combined and dried by passing through a glass funnel containing anhydrous sodium sulfate. The organic part was concentrated by vacuum evaporator and OCPs were analyzed on GC/ECD device. 2.4.3. Determination of OCPs in sediment samples 20g dry sediment was extracted Soxhlet with 300 mL of a mixture of n–Hexan : acetone (1:1) for 16 hours. The extract will be concentrated and made up 10 mL. 5 mL of the extract was purified on an activated florisil packing column (with the length 40 cm and the dimension 2 cm). The elution was conducted with 120 mL of a mixture of n-hexan : DCM (4:1) to obtain OCPs. The extract was concentrated and washed off the colorant and humus by acid (if needed). Finally, the extract was evaporated to 1 ml and stored in the sample vial, OCPs were analyzed on GC/ECD. 2.4.4. Determination of OCPs in biological samples The process of treating biological samples for OCPs analysis was quite similar to that of sediment samples. However, in biological samples, the lipid content was often large, so the washing process with concentrated sulfuric acid was repeated more times (5 times). Also, with biological samples, no additional copper chips were required to remove sulfide compounds. 2.5. Experimental methods on oyster and medaka embryo-larvae 2.5.1. Oyster embryo-larvae DDT 100 ppm was added to the sediments at the content of 2; 10; 20; 50; 200; 1000 µL, respectively, along with the ratio of artificial seawater: sediment (1: 4), the mixture was stirred for 5 minutes, shaken for 8 hours and settled overnight to decant the water. 20 mL of embryonic and larvae solution of oysters were added with 1 mL of DDT solution at different concentrations, each concentration was repeated 3 times. 2.5.2. Medaka embryo-larvae The healthy embryos (embryos with transparent structure, intact embryo membrane, evenly dense yolk mass) was selected and transferred to the experimental well according to the corresponding concentrations of DDT: 0.04; 0.08; 0.12; 0.16; 0.2; 0.24; 0.28 µg/L and control sample (0 µg/L). Each experiment was repeated three times and each well had 10 embryos/concentration.
3 2.6. Methods of toxicity assessment 2.6.1. Determination of LC50, EC50, and mortality 2.6.2. Method QRT-PCR analysis to assess the impact of pesticides on Medaka chemical level molecular biology 2.6.3. Methods of observing the morphology and structure of cells 2.7. Statistical analysis CHAPTER 3: RESULTS AND DISCUSSION 3.1. Group for the sampling locations The sampling sites were classified into two groups based on the similarity of the concentration of 06 OCPs in water and sediments by cluster analysis (CA). The twelve sampling sites were grouped into two clusters (Figure 3.1). Figure 3.1. Cluster analysis in spatial sampling locations 3.2. the State of OCPs in water and sediments 3.2.1. Physical and chemical parameters in surface water and sediments When compared with the National Standard 08 – MT: 2015 / BTNMT column A1 on surface water quality, all water quality parameters (except turbidity) in the Saigon-Dong Nai estuary were within permissible limits. Changes in chemical and physical properties in sediments were influenced mainly by structural factors, for example, sediment types, texture, parent materials, and topographic factors. During the dry season, the high physical and chemical value in sediments was mainly related to low water flows, resulting in high rates of sediment deposition. Sediments collected from the river branches had a higher degree of physical and chemical parameters than those collected from the mainstream of the Saigon - Dong Nai estuary. 3.2.2. The concentration of OCPs in water 3.2.2.1. Fluctuation by seasons The seasonal variation in the concentration of OCPs in the water was largely dependent on the amount of precipitation that moved pollutants from the upstream or surrounding areas. This may deposit the pollutants
4 in downstream areas of rivers, resulting in the OCPs concentration in downstream reaches of the river in the rainy season higher than that in the dry season (Table 3.3). Table 3.3. The concentration of OCP (µg/L) in water in two seasons Dry season Rainy season QCVN 08- OCPs Min-max Mean Min-max Mean MT:2015/BTNMT Total DDTs 0.022–0.3 0.137 0.021–1.42 0.301 Total HCHs 0.022–0.37 0.107 0.068–0.74 0.292 Aldrin KPH–0.065 0.008 0.02–0.133 0.068 Appendix 3 Heptachlor 0.002–0.031 0.009 0.004–0.25 0.040 Dieldrin KPH–0.09 0.007 KPH–0.172 0.024 Endrin 0.007–0.036 0.019 0.004–0.12 0.027 3.2.2.2. Changes by spatial variation (by groups) The concentrations of DDTs, HCHs, aldrin, heptachlor, and dieldrin in water of group 1 were significantly higher than those of group 2 (Table 3.7), showing the effects of agricultural activities. Table 3.7. Concentrations of OCPs (µg/L) in water of the two groups. Cluster 1 Cluster 2 QCVN 08- OCPs Min-max Mean Min-max Mean MT:2015/BTNMT Total DDTs 0.13–1.42 0.46 0.02–0.54 0.139 Total HCHs 0.11–0.75 0.34 0.02–0.51 0.151 Aldrin 0.005–0.13 0.06 KPH–0.1 0.029 Appendix 3 Heptachlor 0.006– 0.07 0.04 0.002–0.07 0.018 Dieldrin 0.006–0.17 0.04 KPH–0.07 0.008 Endrin 0.008–0.12 0.03 0.03–0.11 0.021 3.2.3. The concentration of OCPs in sediment 3.2.3.1. Fluctuation by seasons Residues of OCPs found in sediments were similar to those found in water samples, the concentrations in the rainy season were significantly higher than that in the dry season (Table 3.10). Table 3.10. Concentrations of OCPs (µg / kg) in sediments of the two seasons Dry season Rainy season OCPs QCVN 43:2017/BTNMT Min-max Mean Min-max Mean DDTs 0.09–9.75 3.4 1.22–23.17 8.04 HCHs 0.61–5.66 2.29 1–13.15 4.51 Aldrin KPH–1.68 0.40 KPH–8.96 1.52 Appendix 7 Heptachlor KPH–3.44 1.01 0.22–24.9 3.58 Dieldrin KPH–2.2 0.54 KPH–1.42 0.32 Endrin KPH–2.51 0.97 0.19–4.97 1.40
5 3.2.3.2. Changes by spatial variation (by groups) For sediment, the concentrations of DDTs 11.8 µg/kg, HCHs 6.20 µg/kg, aldrin 2.37 µg/kg, heptachlor 5.94 µg/kg, dieldrin 0.93 µg/kg and endrin 1.64 µg / kg in group 1 were much higher those in group 2 with the concentration of 3.75; 2.47; 0.49; 1.08; 0.26 and 1.03 µg/kg, respectively (Table 3.14). Endrin concentrations in sediments did not differ much between the two groups. Table 3.14. Concentrations of OCPs (µg / kg) in sediment in the two groups Cluster 1 Cluster 2 QCVN 43:2017/BTNMT OCPs Min-max Mean Min-max Mean DDTs 4.6–23.17 11.8 0.09–8.08 3.76 HCHs 2.55–13.15 6.20 0.61–5.52 2.47 Aldrin 0.38–8.96 2.37 KPH–2.67 0.49 Appendix 7 Heptachlor 0.54–24.9 5.94 KPH–3.86 1.08 Dieldrin KPH–2.2 0.93 KPH–1.61 0.26 Endrin 0.19–3.92 1.64 KPH–2.56 1.03 3.2.4. Correlation between the concentration of OCPa in water and sediment The seasonal change may reflect the higher correlation between the concentration of total DDTs and total HCHs in sediment and water in the rainy season than that in the dry season (Figure 3.3). Figure 3.3. The correlation of DDTs and HCHs concentrations in water and sediment Figure 3.4. The correlation of aldrin, heptachlor, dieldrin, and endrin concentrations in water and sediment An increase in the concentration of aldrin in sediments resulted in a significant increase of the aldrin concentration in the water during the rainy season, but not during the dry season (Figure 3.4a). In contrast, the concentrations of heptachlor and endrin in the water also increased markedly along with the increase in sediment concentration during the dry season but not in the rainy season (Figures 3.4b and 3.4d). There was no significant correlation in dieldrin concentrations between water and sediments in the two seasons (Figure 3.4c).
6 3.2.5. Evaluation for the source of OCPs pollution by analyzing the main components PCA / FA was applied to extract three main components (PC) with specific values greater than 1 per season and for each group. The first three OCPs, with three maximum variances considered as VF (latent factors), had eigenvalues greater than 1, cumulatively accounted for 75% of the total variance in the dry season and 84% during the rainy season, respectively, and 87.6% for group 1, and 69.9% for group 2, respectively (Table 3.19). Table 3.19. Correlation of OCPs with latent factors (VF) from PCA/FA analysis in two seasons and two groups Dry season Rainy season Cluster 1 Cluster 2 Parameters VF1 VF2 VF3 VF1 VF2 VF3 VF1 VF2 VF3 VF1 VF2 VF3 Water DDTs 0.53 0.67 0.10 0.53 0.36 0.67 0.70 0.26 0.57 0.18 0.77 -0.25 HCHs 0.18 0.85 0.19 0.46 0.74 0.26 0.67 0.69 0.12 0.71 0.43 -0.20 Aldrin -0.15 0.80 0.36 0.16 0.91 -0.14 0.30 0.90 -0.07 0.87 0.16 -0.18 Heptachlor 0.28 0.62 -0.25 0.86 0.15 0.10 0.87 0.19 0.05 0.65 0.03 -0.08 Dieldrin 0.25 0.20 0.76 0.20 0.15 0.89 0.18 0.11 0.92 0.07 0.74 0.05 Endrin 0.37 0.73 -0.12 -0.23 -0.08 0.88 -0.20 -0.04 0.90 -0.03 0.92 0.04 Sediment DDTs 0.90 0.34 0.08 0.57 0.58 0.48 0.73 0.43 0.43 0.68 0.47 0.35 HCHs 0.83 0.23 -0.06 0.32 0.87 0.21 0.45 0.72 0.17 0.88 0.03 0.29 Aldrin 0.93 0.24 0.01 0.74 0.53 0.19 0.93 0.28 0.07 0.70 0.17 0.40 Heptachlor 0.81 0.19 -0.08 0.88 0.31 0.15 0.93 0.15 -0.05 0.46 0.61 0.41 Dieldrin 0.86 0.00 0.25 0.80 0.24 0.06 0.54 -0.72 -0.10 0.00 -0.07 0.85 Endrin 0.60 0.15 -0.58 0.88 0.25 -0.15 0.97 0.02 -0.07 0.57 -0.37 0.22 Eigenvalue 5.91 1.98 1.11 6.92 2.04 1.18 6.62 2.19 1.69 4.86 2.27 1.26
7 % total 49.2 16.5 9.2 57.6 17.0 9.9 55.2 18.3 14.1 40.5 18.9 10.5 variance Cumulative 49.2 65.8 75.0 57.6 74.6 84.5 55.2 73.5 87.6 40.5 59.4 69.9 percentage variance Note: Bold numbers are those greater than 0.75, and underlined numbers are those greater than 0.5 and smaller than 0.75; VF = varimax factor Major component analysis and factor analysis (PCA/ FA) were used to identify potential constituents in six tested OCPs in water and sediment to identify sources of pollution that could emit these components. The pollution points of PCA are shown in Figure 3.5, the variables generated by the concentration of OCPs mainly at different sampling locations. Figure 3. 5. Two OCPs were extracted when performing PCA / FA analysis for the entire data PC1 accounted for 66.6% and PC2 accounted for 15.2% of the total variance. The variance of OCPs in water and sediment of 12 study sites in the dry season were lower than those in the rainy season. In the dry season, the PC2 value was negative and in the rainy season, the PC2 value was positive. In the rainy season, group 2 has the greatest variance of OCPs concentration. The study results showed that residues of OCPs were detected in most of the water and sediment samples collected at the estuary of the Saigon - Dong Nai River. Therefore, OCPs have the potential to accumulate toxicity in aquatic species in river basins such as fish and bivalve mollusks. 3.3. OCP in fish and bivalve mollusks 3.3.1. The concentration of OCPs in the organism by species 3.3.1.1. Total OCPs OCPs concentration fluctuated with sampling sites, being lowest at ST1 and highest at ST8 in all species examined. The concentration of OCPs found on cockle reached the highest value compared to other species, with values ranging from 6,360 - 45,904 µg/kg (averaged 34,108 µg/kg), followed by goby, clams, green mussels, clam, oysters, respectively: ranged from 7,685 - 40,297 µg/kg (averaged 19,519 µg/ kg); 4,794 -
8 37,585 µg/ kg (average 19,212 µg / kg); 0.323 - 35.359 µg / kg (average 14,320 µg / kg); 7,181–18,462 µg / kg (average 12,376 µg / kg) and 3,007 - 17,081 µg / kg (average 9,297 µg/ kg) (Figure 3.6). Figure 3.6. The concentration of OCPs in fish and bivalve mollusks 3.3.1.2. Total HCHs and isomers Figure 3.7. The concentration of total HCHs in fish and bivalve mollusks Notice: n = 13; a,c: statistically significant difference (5%) on Tukey HSD test The level of HCHs residue of mussel tissue and blood cockle accounted for higher levels than that of the other 4 species. The HCH concentration in mussel tissue was the highest (5,645 µg/kg) and in oysters the lowest (2,702 µg / kg) (Figure 3.7). Isomers α–, β–, γ– and δ – HCH were present in most of the samples collected, and the ratio of β – HCH to total HCHs was highest in many samples. The results also showed that all isomers of HCHs were present in the Saigon-Dong Nai estuary areas. For biological tissues, β – HCH was the dominant isomer and contributed 37-50% to the total value of HCHs observed in different tissues, followed by α–, γ–, δ – HCH, ranged 15 - 32%, 11 - 28% and 9 - 28%, respectively.
9 3.3.1.3. Total DDTs and isomers Significantly different concentrations of DDTs were found in fish and bivalve mollusks, with averaged concentrations of DDTs varying from 3,588 to 9,524 µg/kg. DDTs in collected fish and bivalve mollusks tended to decrease in the order: goby> clam> cockle> green mussel> clam> oyster (Figure 3.9). In terms of data, there was a difference between samples, but through ANOVA analysis, there was no difference in DDTs content in biological samples. This result could be attributed to the different habitats, feeding habits, and their position in the nutritional hierarchy. The concentration of DDTs on goby fish was highest because they live in the bottom, often bury themselves in the mud during the day. Therefore, DDTs could be accumulated with time to increase their concentration. For green-lipped mussels, clams and oysters can cling to the rocky banks, so the capacity to accumulate DDTs was less than that of goby. Figure 3. 9. The concentration of total DDTs in fish and bivalve mollusks Notice: n = 13; a,c: statistically significant difference (5%) on Tukey HSD test The ratios of p, p'– DDD to total DDTs in some species such as goby, oysters, cockles, and clams were dominant, while the ratio of p, p'– DDT in some species such as green mussel and Clam was relatively high. 3.3.1.4. Endosulfans Figure 3.11. The concentration of total endosulfans in fish and bivalve mollusks Notice: n = 13; a,b: statistically significant difference (5%) on Tukey HSD test
10 When fish and bivalve mollusks were exposed to endosulfans, blood cockles accumulated at a significantly higher endosulfans concentration compared to goby, oysters, green mussels, clams and clams (Figure 3.11). ANOVA analysis results showed that the endosulfans content in cockle samples was different from other organisms (p < 0.0001). 3.3.1.5. Other OCPs (heptachlor, aldrin, dieldrin, endrin) Figure 3.12a shows that the cockle sample has the highest heptachlor content, followed by clams, green mussels, clams, goby fish. The difference in concentrations of this toxin accumulated in the six species was quite high. The difference between the cockle samples from 0.453 to 3.032 µg / kg compared with the lowest value in the sample of oyster meat tissue 0.484 µg/ kg, ranging from 0.06 - 1.006 µg / kg. The heptachlor content was relatively low, indicating that bivalve mollusks were not affected significantly. The residual contents of different species were different at p = 0.018, through the results of the post-ANOVA analysis of cockle and oyster samples. There were differences with probability values at 0.0068 and 0.0496, respectively. The differences in heptachlor concentrations between species were statistically significant (Figure 3.12a). Figure 3.12. The concentrations of heptachlor, aldrin, dieldrin, and endrin in fish and bivalve mollusks Notice: n = 13; a,c: statistically significant difference (5%) on Tukey HSD test The concentration of aldrin and endrin detected on the cockle reached the highest value, varying from KPH - 5,421 µg/kg and KPH - 7,104 µg / kg, respectively (Figure 3.12b and 3.12d). The lowest aldrin concentration
11 was on clam with values ranging from KPH - 0.031 µg / kg (average 0.011 µg / kg). Dieldrin concentrations were highest in goby, followed by cockles and lowest in oysters, with mean values 1,743 µg / kg; 1,227 µg / kg and 0.077 µg / kg, respectively (Figure 3.12c). The variation in the content of aldrin and dieldrin among biological samples were not too high. The dieldrin concentration was higher than aldrin, because aldrin is easily converted to dieldrin in the environment. The aldrin concentration was influenced by different species factors. The concentration of aldrin and endrin of blood cockle samples compared to those of the samples of green mussel and clam differed statistically; the probability value was less than 0.0001. The concentration of aldrin and endrin of the samples of clams, goby gobies, and oysters, although different in data, differed statistically, the mean value was the same as that of the samples of cockles, green mussels, and clams, p = 0.0012. There was still a difference in dieldrin content between organisms with a probability value p = 0.0042. Through post-ANOVA control, the concentration of aldrin and endrin of the samples of goby, green mussel, oyster, and clam were different, p
12 The concentrations of OCPs on the sub-river sites were higher than those of the main river due to the intake of different sources of pollution from the sub-tributaries at the Saigon-Dong Nai estuary. They flowed through areas of widespread agricultural activity where insecticides were widely used so contaminants entered into estuarine water. Due to the persistence and toxicity of OCPs in the environment, OCPs were prohibited or controlled for use in agricultural operations. 3.3.3. Source of contamination by OCPs in organisms Key component analysis and factor analysis (PCA/ FA) were used to identify potential constituents in the seven tested OCPs in biological tissues and to identify possible sources of intrusive contamination for these ingredients. PCA / FA was extracted into two main components (PC) with eigenvalues greater than 1. Corresponding maximum variance (VF) (latent factor) has eigenvalues greater than 1, cumulatively accounted for 64.7 % of total variance value (Table 3.23). The first factor explained 46.7% of the total variance and showed a high load for DDTs, aldrin, and dieldrin, as well as a moderate load for HCHs and endrin. The second factor was characterized by a high positive load for endosulfans and a medium load for heptachlor and endrin, which accounted for 18% of the total variance. Table 3. 23. Loading of OCPs on different latent factors formed from PCA/FA analysis Parameters VF1 VF2 Total HCHs 0.56 0.41 Total DDTs 0.77 -0.07 Heptachlor 0.26 0.58 Aldrin 0.85 0.29 Diedrin 0.86 0.13 Endrin 0.52 0.66 Total_endosulfan -0.18 0.86 Eigenvalue 3.27 1.26 % total variance 46.7 18.0 Cumulative percentage variance 46.7 64.7 Note: Bold numbers are those greater than 0.75, and underlined numbers are those greater than 0.5 and smaller than 0.75; VF = varimax factor As a result, PC1 explained 46.7% and PC2 explained 64.7% of the total variance (Figure 3.15). The different distributions of fish and bivalve molluscs along PC1 and PC2 in the PCA plots indicated that these variables may explain the pattern of OCPs found. Two main factors were used to categorize the pollutants based on concentrations of OCPs. Analysis results showed that cockle samples had a much wider range of OCPs than those of other species and OCPs concentration of mussels was the lowest.
13 Figure 3. 15. Grouping of fish and bivalve mollusks based on principal component analysis/factor analysis 3.4. Assessment of the toxicity of DDT The results of assessing the concentrations of OCPs in water, sediments, and organisms in the Saigon - Dong Nai estuary showed that DDT was the chemical that accounted for the highest concentration and mainly in the collected samples. In addition, DDT is a common chemical used in agriculture to prevent insect infestation on plants and to kill many insects that cause epidemics for humans. Due to its toxicity and popularity in the environment, chemical DDT was selected to evaluate the toxicity on aquatic organisms and embryos. 3.4.1. Toxicity of DDT on the growth of embryos and Pacific oyster larvae 3.4.1.1. Survey in water Figure 3. 16. Net percentages of abnormal development (±SE) in C. gigas embryo observed after 2h of exposure to DDT in an artificial seawater environment
14 DDT greatly affected the developmental ability of Pacific oyster embryos after 2 hours of exposure to artificial seawater. The rate of the slow-growing embryo, undifferentiated linear changed linearly with an increase of DDT concentration. The rate of delayed embryo growth increased from 28% to 58%, corresponding to a concentration of 0.1 to 100 µg/L, compared to that of the control sample (2%) (Figure 3.16). The effect of DDT on growth retardation of oyster embryos after 2 hours of exposure in water was established with EC50 value of 66.88 µg / L. Figure 3. 18. Net percentages of mortality (±SE) in C. gigas embryo observed after 24 h of exposure to DDT in an artificial seawater environment The mortality rates of embryos and larvae changed linearly with the increasing concentration of DDT in the water environment. Mortality rates varied from 44% to 69%, corresponding to an increase in DDT exposure from 0.1 to 100 µg/L, compared to those of the control sample 3% (0 µ g/L ( Figure 3.18). The effect of DDT on mortality of embryos and oyster larvae after 24 hours of exposure in water was established with LC50 value at 4.62 µg/L. 3.4.1.2. Survey in sediments In the control sample, the rate of growth retardation was 2%, in the experimental samples, this rate increased from 18% to 75% linearly with the increase in DDT concentration from 0.01 to 5 mg / kg after 2 hours of exposure (Figure 3.20). The effect of DDT on embryo growth retardation after 24 hours of exposure in sediments was established with EC50 value at 1.1 mg/kg.
15 Figure 3. 20. Net percentages of abnormal development (±SE) in C. gigas embryo observed after 2h of exposure to DDT in sediment environment Figure 3. 22. Net percentages of mortality (±SE) in C. gigas embryo observed after 24 h of exposure to DDT in sediment environment Mortality rates of embryos and oyster larvae in the control samples were quite low, only 3% compared to those of the experimental samples (from 27% to 84%), corresponding to an increase in DDT exposure concentrations from 0.01. up to 5 mg/kg (Figure 3.22). The effect of DDT on mortality of embryos and oyster larvae after 24 hours of exposure in sediments was established with LC50 value at 0.3 mg/kg. 3.4.1.3. Investigation of embryonic morphology and oyster larvae • In water
16 Figure 3. 24. Scanning Electron Microscopy (SEM) micrograph of C. gigas in artificial seawater environment at 24h Figure 3. 25. Transmission Electron Microscope (TEM) images illustrating the ultra structural changes C. gigas in artificial seawater environment (control sample, without DDT) after 24h SEM scan results showed that oyster embryos in the control sample were round or spherical shape with a smooth, slick outer surface and the cell division was underway (Figure 3.24a). The embryos of oysters became deformed and their appearances were rough and broken after being exposed to the pesticide DDT (Figure 3.24b, c, d). This proved that pesticides have significantly changed the embryo morphology and even killed embryos.
17 In the control sample (Figure 3.25a, b, c, d), TEM images at different positions showed that without the addition of pesticides DDT and culture of embryos under normal conditions, the embryonic hyperstructure oysters were spherical or circular (Figure 3.25d), with a well-defined internal organelle. Inside the cytoplasm, the fully intact endoplasmic reticulum (arrow 2) and the granules have intact capsids and mitochondria (arrow 1), a dense, distinct internal nucleus below the accessory muscle layer (Figure 3.25a), c); the outermost cell wall of the embryo was thick (measured size 610 nm, Figure 3.25b). After 24 hours of exposure to the pesticide DDT at a concentration of 1 g/L, the internal organs were almost destroyed (Figure 3.26b, c, d). The cell wall was thinner (405-440 nm, Figure 3.26a) and the capsids with the inner nucleus were destroyed and empty (arrow Figure 3.26b); the endoplasmic reticulum was not intact (arrow Figure 3.26d). This proved that the pesticide DDT has affected the organelle structure inside the oyster embryo. Figure 3. 26. Transmission Electron Microscope (TEM) images illustrating the ultra structural changes of C. gigas embryo in artificial seawater environment (the exposure DDT concentration of 1g.L-1) after 24h • In sediments Similar to the artificial seawater environment, the SEM image of the surface structure of the Pacific oyster embryo C. gigas in the sediment samples and the control samples (not exposed to DDTs, Figure 3.27a) and the experimental samples (exposure infection with DDTs 1 mg/kg, Figure 3.27b, c, d) also differed significantly. In the control samples, the surface structure of the oyster embryo was smooth and slick and undergoing cell division (Figure 3.27a). In contrast, in the experimental samples, the oyster embryo surface structure was strongly affected, the embryo was strongly destroyed, even broken, causing the death of the embryo (Figure 3.27b, c, d).
18 Figure 3. 27. Scanning Electron Microscopy (SEM) micrograph of C. gigas in the sediment environment at 24h Figure 3. 28. Transmission Electron Microscope (TEM) images illustrating the ultra structural changes C. gigas in sediment environment (control sample, without DDT) after 24h