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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 10, 2014 to January 5, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & Chinese test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 211 Daphnia magna Reproduction Test [M]. Second edition. Beijing: China Environmental Press. 2013: 94 - 112.
Deviations:
no
Qualifier:
according to
Guideline:
other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 21828 - 2008, Chemicals Daphnia magna Reproduction Test [S]. Beijing; China Standards Press, 2008.
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration of China. HJ/T 153-2004, The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.
GLP compliance:
yes
Specific details on test material used for the study:
Physicochemical properties
Melting point: -39 °C at 101.3 kPa
Boiling point: 216 °C at 101.3 kPa
Volatility: Non-volatile
Vapour Pressure: 1.22 kPa at 37.8 °C
Solubility in Water: Using the US EPA On-Line EPI Suite™ WSKOW v1.42 model, the water solubility range is predicted to be 2.64E-27 to 0.004421mg/L. This indicates that the substance is insoluble, with water solubilities much lower than 0.1mg/L. Solubility in Solvent Water: none Density: 0.82 Odor: None
Analytical monitoring:
yes
Details on sampling:
The actual concentrations of the nominal loading rate of 100mg/L WAF test group were measured at 0d, 5d, 9d, 13d, 16d, 20d (new solution) and 1d, 6d, 10d, 14d, 17d, 21d (old solution) by GC. The actual concentrations of blank control were measured at 0d and 1d (before the renewal) after the start of the test.
Vehicle:
no
Details on test solutions:
0.500g test substance was obtained in a flask with a small amount of test medium then transferred into 5L-jar and diluted with test medium to 5000mL. And the test solution was stirred with a magnetic stirrer for about 24h (the depth of vortex was maintained in the range of 2cm-3cm), then the nominal loading rate of 100 mg/L suspension was obtained. After the stirring was stopped, the mixture was allowed to stand for about 1 hour. Then the aqueous phase from the middle and bottom layer was removed by siphoning into a beaker (the first approximated 100-200mL discarded) to give the nominal loading rate of 100mg/L WAF (Water-accommodated fraction) solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna Reason for selection of species: The test species is recommended by "The Guidelines for the Testing of Chemicals" Supplier: The parent animals were supplied by Safety Evaluation Center, Shenyang Research Institute of Chemical Industry. Apparently healthy, and bred in our laboratory. The test animal batch number was DM20130801-1. Acclimation: At the start of the test, Daphnia magna were acclimated in the testing laboratory for one generation at least under the same conditions of water quality (Elendt M4 medium), temperature (20°C ± 2°C), light and dark period (16h light / 8h dark), illumination intensity (600 lx -1200 lx). During the acclimation, Daphnia magna were fed with a concentrated algal suspension. Size: The animals were less than 24 hours old and were not first brood progeny. The female Daphnia magna were used in the test. Number: 20 Allocation to the test groups: The animals were allocated at random to each test group.Food Species: Scenedesmus obliquus Supplier: Committee on Type Culture Collection of Chinese Academy of Sciences. Condition of culture: Temperature (23°C ± 2°C), 24h continuously light, illumination intensity (4440 lx~8880 lx).
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
During of the test, hardness was 230mg/L - 240mg/L.
Test temperature:
During the test, the result of continuous monitoring of the water temperature was shown that the water temperature was 19.4°C - 20.2°C.
pH:
During of the test, pH of test solutions was 8.10 - 8.62
Dissolved oxygen:
During of the test, DO was 8.7mg/L - 9.4mg/L
Salinity:
Not applicable - Freshwater study
Nominal and measured concentrations:
The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0d in the new solution and at 1d in the old test solution after the start of the test.The measured concentrations of test substance in 100mg/L loading rate WAF test group were all lower than the limit of detection (LOD = 0.863 mg/L) at 0d, 5d, 9d, 13d, 16d, 20d in the new solution and at 1d, 6d, 10d, 14d, 17d, 21d in the old solution.The actual concentrations of the nominal loading rate of 100mg/L WAF test group were measured at 0d, 5d, 9d, 13d, 16d, 20d (new solution) and 1d, 6d, 10d, 14d, 17d, 21d (old solution) by GC. The actual concentrations of blank control were measured at 0d and 1d (before the renewal) after the start of the test.
Details on test conditions:
Instruments: 150mL beaker (test vessel), 2000mL Flask with a ground-in stopper, dropper, pipette, transferpettor, measuring cylinder, volumetric flask and so on. Test methodsPreparation of the test solution of the test substance: 0.500g test substance was obtained in a flask with a small amount of test medium then transferred into 5L-jar and diluted with test medium to 5000mL. And the test solution was stirred with a magnetic stirrer for about 24h (the depth of vortex was maintained in the range of 2cm-3cm), then the nominal loading rate of 100 mg/L suspension was obtained. After the stirring was stopped, the mixture was allowed to stand for about 1 hour. Then the aqueous phase from the middle and bottom layer was removed by siphoning into a beaker (the first approximated 100-200mL discarded) to give the nominal loading rate of 100mg/L WAF (Water-accommodated fraction) solution. Preparation of the test solution: Elendt M4 medium was divided into 10 test vessels, respectively, and the volume of test medium is 80mL each test vessel as blank control group. The nominal loading rate of 100mg/L WAF test solution was divided into 10 test vessels, respectively, and the volume of test medium is 80 mL each test vessel as test substance group. Test conditions Conditions of exposure Type of test: The measured concentrations of the test substance in the solution were all lower than the limit of detection (LOD=0.863mg/L) during 24h, and the stability of test substance in test system could not be confirmed, so the test was conducted as semi-static test at 24h intervals. Exposure duration: 21d Test concentration: According to the results of other tests, a nominal loading rate of 100mg/L water-accommodated fraction (WAF) solution test group. Control: A control (test medium without test substance) was run in parallel to the test concentrations. Replicate: 10 replicates Number of animal: 10 / test groupVolume of test solution: 80mL / test vessel Conditions of test environment Water temperature: 20℃ ± 2℃, the total variability should be < 2℃. Dissolved oxygen concentration: The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test should be kept at or above 3 mg/L. pH: The test was performed without adjustment of pH. The pH of the test medium should be in the range of 6.0 to 9.0, and vary by less than 1.5 units during the test. Light: Artificial light of white fluorescent lamp, 16h light / 8h darkness, illumination intensity was 600 lx -1200 lx.Feeding: The animals were fed with a concentrated algal suspension daily during the exposure. The diet of the parent animals was 0.1mg C/Daphnia/day - 0.2mg C/Daphnia/day. Observation and measurements Vortex depth measurements: The vortex depth was measured at the start and end of the mixing period and the appearance of vortex were also recorded. Observations on test substance solubility: At the start and end of each mixing period, and after the standing period the appearance of the WAF solution was observed and recorded. After siphoning, the WAF solution was inspected under microscope. Observation of parent daphnias and offspring: The number of dead parent daphnias was recorded daily. The live offspring produced by each parent animal was removed and counted daily, and also the presence of dead offspring and aborted eggs were recorded. The body length of parent daphnias: At the end of the test, the body length of each surviving parent daphnias (anal spine not included) was determined using digital caliper (The precision was 0.01mm). Measurement of intensity of illumination and temperature, pH, dissolved oxygen, total hardness of test solution: The intensity of illumination of test environment was measured at the start and the end of the test, respectively.The water temperature, pH, DO and hardness of blank control and the nominal loading rate of 100mg/L WAF test group were measured in the new test solution on day 0, day 1, day 8, day 9, day 13, day 14, day 18 and day 19. The water temperature, pH, DO and hardness of blank control and the nominal loading rate of 100mg/L WAF test group were measured in old test solutions on day 1, day 2, day 9, day 10, day 14, day 15, day 19 and day 20. Measurement of the actual concentrations of the test substance in the test solutions: The actual concentrations of the nominal loading rate of 100mg/L WAF test group were measured at 0d, 5d, 9d, 13d, 16d, 20d (new solution) and 1d, 6d, 10d, 14d, 17d, 21d (old solution) by GC. The actual concentrations of blank control were measured at 0d and 1d (before the renewal) after the start of the test.Data processing: Data were analyzed by appropriate statistical methods to calculate the mean reproductive output across replicates for each concentration and the pooled residual standard deviation.For the estimation of the “LOEL” and the “NOEL” the numbers of live offspring produced per adult and daphnia length data over the duration of the test for the control and each test group were compared using T-test with Bonferroni adjustment for comparing several treatments with a control. The statistical analyse was performed using the SPSS computer software (Statistical Product and Service Solutions) (edition 16.0).
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
other: LOEL
Effect conc.:
> 100 other: loading rate mg/L WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: LOEL
Effect conc.:
> 100 other: loading rate mg/L WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
body length
Duration:
21 d
Dose descriptor:
other: LOEL
Effect conc.:
> 100 other: loading rate mg/L WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Parent animal
Duration:
21 d
Dose descriptor:
other: NOEL
Effect conc.:
100 other: loading rate mg/L WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: NOEL
Effect conc.:
100 other: loading rate mg/L WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
body length
Duration:
21 d
Dose descriptor:
other: NOEL
Effect conc.:
100 other: loading rate mg/L WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parent animal
Details on results:
Vortex depth and appearance measurements: The vortex depth was recorded at the start and end of each mixing period. And the vortex was observed to be a dimple at the water surface on each occasion at the start and end of each mixing period. Observations on test substance solubility: At the start and end of each mixing period, the 100mg/L loading rate suspension was observed to be water column with oily globules of test substance floating on the water surface. After 24h stirring and 1-hour standing period, the 100mg/L loading rate suspension was observed to remain a clear colorless water column in the mid-depth but with oily globules of test substance floating on the water surface. After siphoning, the microscopic inspection of WAF solution showed no micro-dispersions or undissolved test substance to be present. Mortality rate of parent animals: At the end of the test, there were no parent animal dead in the control group, which met the quality requirement that the mortality of the parent animals (female Daphnia) should not exceed 20% at the end of the test. At the end of the test, the mortality of parent animals in the nominal loading rate of 100mg/L WAF test group was 0%. The results revealed that there was no statistically significant differences (p>0.05) between the control and the nominal loading rate of 100mg/L WAF test group in terms of the mortality of parent animals. So according to the mortality of parent animal, LOEL was greater than nominal loading rate of 100mg/L WAF, NOEL was nominal loading rate of 100mg/L WAF.Living offspring by each parent animal: At the end of the test, the mean ± SD of reproductive output of the control group was 148±20 offspring per surviving parent animal. The coefficient of variation in the control group was thus 14%. Offspring were first produced in the blank control on day 8 of the test.No dead offspring and aborted broods were observed in blank control and nominal loading rate of 100mg/L WAF test group during the test. The total number of living offspring produced per surviving parent animal in each concentration of the test substance were estimated by T-test with Bonferroni adjustment. The results revealed that there were no statistically significant differences (p>0.05) between the control and the nominal loading rate of 100mg/L WAF test group in terms of the number of living offspring produced per adult. So according to the reproductive output, LOEL was greater than nominal loading rate of 100mg/L WAF, NOEL was nominal loading rate of 100mg/L WAF. Body length of the parent animals: At the end of the test, there were no statistically significant differences (p>0.05) between the means for nominal loading rate of 100mg/L WAF test group and the control in terms of body length by T-test with Bonferroni adjustment. So according to the body length of the parent animals, LOEL was greater than nominal loading rate of 100mg/L WAF, NOEL was nominal loading rate of 100mg/L WAF.Measurements of water temperature, pH, DO and hardness The intensity mean values of illumination at the start and the end of the test were 683 lx and 685 lx, respectively. During the test, the result of continuous monitoring of the water temperature was shown that the water temperature was 19.4°C - 20.2°C. The water temperature, pH, DO and hardness of blank control and the nominal loading rate of 100mg/L WAF test group were measured in new test solutions on day 0, day 1, day 8, day 9, day 13, day 14, day 18 and day 19 and were measured in old test solutions on day 1, day 2, day 9, day 10, day 14, day 15, day 19 and day 20. During of the test, pH of test solutions was 8.10 - 8.62; DO was 8.7mg/L - 9.4mg/L; hardness was 230mg/L - 240mg/L. Measurement of test substance concentrations The test solutions’ pretreatment and saved: 50mL test solution was accurately taken and poured into the separating funnel, then 20mL, 15mL, 10mL hexyl hydride was added respectively to extract for three times (5min for each time). After that, each extraction was collected and transferred into a 50mL volumetric flask and then diluted with hexyl hydride to 50mL. The actual concentrations of test substance in extractions were measured. The samples were analyzed immediately. Otherwise the samples (hexyl hydride extractions) were stored at 4°C until analyzed during one week.The result of the measurement of test substance concentrations: The results of the analytical method of test substance are shown in the final report of “Validation of An Analytical Method of NovaSpec Base Oil” (The report No. is A14048-T14037-1R). The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0d in the new solution and at 1d in the old test solution after the start of the test. The measured concentrations of test substance in 100mg/L loading rate WAF test group were all lower than the limit of detection (LOD = 0.863 mg/L) at 0d, 5d, 9d, 13d, 16d, 20d in the new solution and at 1d, 6d, 10d, 14d, 17d, 21d in the old solution.
Results with reference substance (positive control):
Reference substance not used in this study.
Reported statistics and error estimates:
Validity of test (1) The animals were less than 24 hours old and were first brood progeny. (2) The mortality of the parent animals (female Daphnia) was 0%, which was not exceed 20% at the end of the test in the control. (3) The mean number of live offspring produced per parent animal surviving was 148 at the end of the test in the control, which was greater than 60. (4) The coefficient of variation around the mean number of living offspring produced per parent animal in the control was 14%, which was ≤ 25%.

Vortex depth and observation measurements at the start and end of mixing period

Measurements

100 mg/L loading rate WAF test group

0d

1d

2d

3d

4d

5d

6d

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Depth of vortex (cm)

3.0

2.7

2.7

2.2

2.9

2.7

2.7

2.8

2.7

2.7

2.9

2.7

2.6

2.7

Observation of vortex

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Vortex depth and observation measurements at the start and end of mixing period (continued)

Measurements

100 mg/L loading rate WAF test group

7d

8d

9d

10d

11d

12d

13d

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Depth of vortex (cm)

2.6

2.6

2.7

2.7

2.6

2.7

2.7

2.4

2.3

2.4

2.5

2.6

2.6

2.4

Observation of vortex

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Vortex depth and observation measurements at the start and end of mixing period (continued)

Measurements

100 mg/L loading rate WAF test group

14d

15d

16d

17d

18d

19d

20d

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Depth of vortex (cm)

2.4

2.4

2.4

2.6

2.5

2.7

2.5

2.3

2.5

2.8

2.3

2.4

2.4

2.5

Observation of vortex

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

 

The total number of living offspring produced per parent animal during the test

Nominal loading rate (mg/L)

Replicate

The number of living offspring produced per parent animal

8d

9d

10d

11d

12d

13d

14d

15d

16d

17d

18d

19d

20d

21d

Total number

Blank control

1

0

8

0

9

27

0

0

44

0

0

18

0

0

31

128

2

0

0

0

20

6

0

0

40

0

0

31

0

0

39

136

3

13

0

0

22

0

0

43

0

0

43

0

0

0

48

169

4

15

0

0

25

0

0

46

0

0

49

0

0

11

35

181

5

1

7

0

13

17

0

1

34

0

0

44

0

0

36

153

6

15

0

0

29

0

0

14

21

0

0

41

0

0

35

155

7

0

0

19

0

0

0

30

0

0

36

0

0

36

0

121

8

3

9

0

21

1

0

0

43

0

0

43

0

0

38

158

9

12

0

0

23

0

0

38

0

0

16

27

0

0

44

160

10

20

0

0

30

0

0

0

38

0

0

35

0

0

0

123

100

1

22

0

0

26

0

0

1

41

0

0

32

0

0

38

160

2

19

0

0

23

0

0

0

38

0

0

47

0

0

41

168

3

19

0

0

25

0

0

42

0

0

45

0

0

0

33

164

4

20

0

0

24

0

0

0

47

0

1

28

0

0

36

156

5

17

0

0

26

0

0

0

40

0

0

47

0

0

40

170

6

0

0

26

0

0

0

33

0

0

31

0

0

43

0

133

7

17

0

0

4

26

0

0

39

0

0

36

0

0

24

146

8

0

0

25

0

0

0

29

0

0

29

0

0

40

0

123

9

0

0

21

0

0

33

0

0

0

30

0

0

28

0

112

10

0

0

20

0

0

34

0

0

0

28

0

0

35

0

117

 

The mortality of parent animal per group at the end of the test

Nominal loading rate (mg/L)

Total

Mortality

Mortality

Blank control

10

0

0%

100

10

0

0%

 

The mean ± SD of living offspring produced per parent animal alive at the end of the test

Nominal loading rate (mg/L)

Mean ± SD

Blank control

148 ± 20

100

145 ± 22

Note: SD = Standard deviation

 

The mean ± SD of body length of parent animal alive at the end of the test

Nominal loading rate (mg/L)

Mean ± SD

Blank control

3.97 ± 0.096

100

3.98 ± 0.078

Note: SD = Standard deviation

 

Measured intensity of illumination at the start and the end of the test respectively

Time

Intensity of illumination (lx)

1

2

3

Mean value

The start

693

685

672

683

The end

639

725

692

685

 

pH, DO and hardness in test solutions

Time

Blank control

Nominal loading rate 100 mg/L WAF

pH

DO (mg/L)

Hardness (mg/L)

pH

DO (mg/L)

Hardness (mg/L)

0d new

8.33

8.7

234

8.21

9.3

230

1d old

8.52

9.2

235

8.39

9.3

230

1d new

8.41

8.9

240

9.32

8.8

235

2d old

8.57

9.3

240

8.47

9.0

240

8d new

8.44

8.7

240

8.36

8.7

240

9d old

8.62

9.3

235

8.56

9.2

240

9d new

8.37

9.0

235

8.22

9.0

235

10d old

8.57

9.1

235

8.31

9.3

235

13d new

8.46

8.9

235

8.27

8.9

235

14d old

8.54

9.2

235

8.42

9.2

235

14d new

8.36

9.0

240

8.34

8.9

235

15d old

8.26

9.3

240

8.10

9.4

235

19d new

8.37

9.2

235

8.24

9.0

235

19 old

8.14

9.3

240

8.12

9.3

235

19d new

8.27

9.0

235

8.27

9.0

235

20d old

8.16

9.4

240

8.14

9.4

235

Validity criteria fulfilled:
yes
Conclusions:
21d Lowest Observed Effect Loading rate (LOEL): LOEL for effects on reproduction Greater than nominal loading rate of 100mg/L WAF
LOEL for effects on body length: Greater than nominal loading rate of 100mg/L WAF
LOEL for mortality of parent animals: Greater than nominal loading rate of 100mg/L WAF
21d No Observed Effect Loading rate (NOEL): NOEL for effects on reproduction Nominal loading rate of 100mg/L WAF
NOEL for effects on body length: Nominal loading rate of 100mg/L WAF
NOEL for mortality of parent animals: Nominal loading rate of 100mg/L WAF
Executive summary:

The primary objective of the test is to assess the effect of chemicals on the reproductive output of Daphnia magna. To this end, young female Daphnia (the parent animals), aged less than 24 hours at the start of the test, are exposed to the test substance added to water at a range of concentrations. The test duration is 21 days. At the end of the test, the total number of living offspring produced is assessed. The reproductive output and growth of the adult daphnias exposed to the test substance is compared to that of the control(s) in order to determine the lowest observed effect loading rate (LOEL) and hence the no observed effect loading rate (NOEL). In addition, and as far as possible, the data are analysed using a regression model in order to estimate the loading rate that would cause a x % reduction in reproductive output, i.e. ELx (e.g. EL50, EL20, or EL10).

The survival of the parent animals and time to production of first brood must also be reported. Other substance-related effects on parameters such as growth (e.g. length), and possibly intrinsic rate of increase, may also be examined.

 

Guidelines

[1] Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 211 Daphnia magna Reproduction Test. Second edition. Beijing: China Environmental Press. 2013: 94 - 112.

[2] OECD Guidelines for the Testing of Chemicals. 211. Daphnia magna Reproduction Test. Paris: OECD, Adopted 2 October, 2012.

[3] China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 21828 - 2008, Chemicals Daphnia magna Reproduction Test. Beijing:China Standards Press, 2008.

[4] OECD series on testing and assessment number 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Paris: OECD, Adopted September 2000.

[5] Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. Designation: D6081 – 98 (Reapproved 2009).

[6] Standard Terminology Relating to Biodegradability and Ecotoxicity of Lubricants. Designation: D6384 – 11

[7] State Environmental Protection Administration of China. HJ/T 153-2004, The Guidelines for the test of chemical. Beijing: China Environmental Science Press.2004.

 

Test conditions

Test substance: NovaSpec Base Oil

Test organism: Daphnia magna

Exposure duration: 21d

Test concentration: According to the results of other tests, a nominal loading rate of 100mg/L water-accommodated fraction (WAF) solution test group and a blank control containing no test substance was selected.

Replicate: 10 replicates

Number of animals: 10 / test group

Test medium: Elendt M4 medium

Type of test: Semi-static test (renewal at every 24h)

Preparation of test solution: 0.500g test substance was obtained in a flask with a small amount of test medium then transferred into 5L-jar and diluted with test medium to 5000mL. And the test solution was stirred with a magnetic stirrer for about 24h (the depth of vortex was maintained in the range of 2cm-3cm), then the nominal loading rate of 100 mg/L suspension was obtained. After the stirring was stopped, the mixture was allowed to stand for about 1 hour. Then the aqueous phase from the middle and bottom layer was removed by siphoning into a beaker (the first approximated 100-200mL discarded) to give the nominal loading rate of 100mg/L WAF (Water-accommodated fraction) solution.

Preparation of test system: Elendt M4 medium was divided into 10 test vessels, respectively, and the volume of test medium is 80mL each test vessel as blank control group. The nominal loading rate of 100mg/L WAF test solution was divided into 10 test vessels, respectively, and the volume of test medium is 80 mL each test vessel as test substance group.

Volume of test solution: 80mL / test vessel

Temperature of test solutions: 19.4°C - 20.2°C

Photoperiod: 16h illumination / 8h darkness

Feeding: A concentrated algal suspension is fed to the Daphnia daily, the supplied diet was 0.1mg C/Daphnia/day - 0.2mg C/Daphnia/day.

Aeration: No aeration

Analysis of the actual concentrations of test substance in test solution: The actual concentrations of the nominal loading rate of 100mg/L WAF test group were measured at 0d, 5d, 9d, 13d, 16d, 20d (new solution) and 1d, 6d, 10d, 14d, 17d, 21d (old solution) by GC. The actual concentrations of blank control were measured at 0d and 1d (before the renewal) after the start of the test.

 

Results

The measured concentrations of the test substance in test solutions: The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0d in the new solution and at 1d in the old test solution after the start of the test.

The measured concentrations of test substance in 100mg/L loading rate WAF test group were all lower than the limit of detection (LOD = 0.863 mg/L) at 0d, 5d, 9d, 13d, 16d, 20d in the new solution and at 1d, 6d, 10d, 14d, 17d, 21d in the old solution.

 

21d Lowest Observed Effect Loading rate (LOEL)

LOEL for effects on reproduction Greater than nominal loading rate of 100mg/L WAF

LOEL for effects on body length Greater than nominal loading rate of 100mg/L WAF

LOEL for mortality of parent animals Greater than nominal loading rate of 100mg/L WAF

 

21d No Observed Effect Loading rate (NOEL)

NOEL for effects on reproduction Nominal loading rate of 100mg/L WAF

NOEL for effects on body length Nominal loading rate of 100mg/L WAF

NOEL for mortality of parent animals Nominal loading rate of 100mg/L WAF

 

Discussion: Due to the low aqueous solubility and complex nature of test substance, so according to the guidelines of “OECD series on testing and assessment number 23, Guidance Document on Aquatic Toxic Testing of Difficult Substances and Mixtures”, the test substance solution was prepared as a Water Accommodated Fraction (WAF). During the test, the chemical analysis results showed that the measured concentration of 100mg/L loading rate WAF was lower than the limit of detection of 0.863mg/L. However, the test substance is a complex mixture, its toxicity cannot be attributed to a single component or a mixture of components, but to the test substance as a whole, the test results were based on nominal loading rate only.

No classification is applicable.

Description of key information

Assessment of chronic toxicity to invertebrates

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
100 mg/L

Additional information

A Klimish 1 GLP study conducted in accordance with current OECD guidelines is available to assess the effects of acute toxicity to Daphnia Magna. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 21-Day Lowest Observed Effect Loading rate value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. This category of substances does not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of the a specific study.

No classification is applicable.