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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:
20 August 2018 to 12 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
other: The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 211 Daphnia magna reproduction test [M].
Version / remarks:
The editorial board of “The Guidelines for the Testing of Chemicals” of Chemical Registration Center of the Ministry of Environmental Protection. Second Edition Beijing: China Environmental Press. 2013, page 94-112.
Deviations:
no
Qualifier:
according to
Guideline:
other: National Standardization Technical Committee for the Management of Hazardous Chemicals. GB/T 21828-2008 Chemicals- Daphnia magna reproduction test [S].
Version / remarks:
Beijing: China Standard Press, 2008.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- The concentration of test material in the solutions was determined using LC/MS/MS and TOC analyser.
- Samples were taken for analysis at the beginning of exposure (0 d), 1, 2, 8, 14 and 20 days of exposure.
- On each occasion, four samples (one sample was retained by cold storage in case reanalysis was required) were taken from test material treatment group and one from blank control group for LC/MS/MS analysis. Since Green algae was used as food for the test organism during the test, which may affect the measurement of TOC concentration. Only the freshly prepared solutions in test material treatment and blank control groups were taken for TOC analysis. On each occasion, two samples were taken from each group.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Test material treatment group: the test solution was prepared by the WAF method. The test material was accurately weighed (the range of test material was 49.6-50.4 mg) and added to 5 L of test water to give the loading rate of 10 mg/L. After the addition of test material, the solutions were stirred by magnetic stirrer (the vortex height was about 14 % of the liquid depth in the test vessel). The stirring was maintained for 24 hours, followed by a settling phase of 24 hours. A siphon sealed by a piece of Parafilm was immersed into the solution avoiding insoluble test material on the surface. And then, an iron wire passed through the siphon from inside and push off the parafilm. The solution was withdrawn from the middle layer (the first 100 mL was discarded) and used for exposure. The test solution was freshly prepared and no micro-emulsions were observed by microscopy before use. Each test vessel was loaded with 80 mL of solution.
- Blank control: the method of preparation was equivalent to that used in the test material treatment group but no test material. Each test vessel was loaded with 80 mL of solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Justification for species: Daphnia magna is recommended as the test species by the Guidelines for the Testing of Chemicals. Characteristics which made this test organism suitable for toxicity testing are its ease of culturing and sensitivity to a variety of chemical substances.
- Source: The species Daphnia magna was originally supplied by Safety Evaluation Centre, Shenyang Research Institute of Chemical Industry. Since that time, the clone has been breeding in CTI-SET.
- Feeding during test: The well concentrated algae suspensions were provided every day, and the supplied diet was between 0.1-0.2 mg C/Daphnia / day.

ACCLIMATION
- The tap water dechlorinated by aeration was used to maintain the animals. During the acclimation, the temperature was controlled within the range 18-22 °C, the pH values were 6-9, and the dissolved oxygen concentration was not less than 3 mg/L. The test organisms, less than 24 hours old and not the first brood from parent daphnids (Daphnia magna C20180806), were selected as test organism.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
160 mg/L (calculated as CaCO3)
Test temperature:
21.2 - 21.7 °C
pH:
7.77 - 8.71
Dissolved oxygen:
8.02 - 8.75 mg/L
Nominal and measured concentrations:
Nominal: 10 mg/L WAF
Geometric mean of measured concentration: 0.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: wide mouth bottle with volume of 125 mL
- Fill volume: 80 mL
- Renewal rate of test solution: As the test material is a UVCB substance, the semi-static exposure regime was used and the test solution was renewed every day, to ensure constant exposure concentrations, as far as possible.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the tap water de-chlorinated by aeration was used as the test water, the TOC should be less than 2 mg/L and hardness should be more than 140 mg/L (calculated as CaCO3)
- Intervals of water quality measurement: The TOC of the test water was measured at the start of the test. The hardness of test solution in blank control and at the loading rate of 10 mg/L were measured at the start of the test. The temperature, pH values and dissolved oxygen of the test solution in control and test material treatment group were measured at the beginning of exposure (0d) and on day 2, 8, 14 and 21 of exposure. One sample was taken from test material treatment and blank control group. The pH values should be within the range 6-9, and it should not vary by more than 1.5 units, the dissolved oxygen concentration was above 3 mg/L. The light intensity of the test area was measured once per week. The light intensity should be in the range of 1000-1500 lux.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour dark
- Light intensity: 1040 - 1360 lux

EFFECT PARAMETERS MEASURED:
- The mortality of the parent animals was observed daily from the beginning of exposure.
- The time to production of first eggs and first brood was recorded from the fourth day of the exposure.
- The offspring produced by parent animal were removed daily after the production of first brood to reduce the food consumption. The living offspring and aborted broods were counted and the presence of males or ephippia was recorded. The coefficient of variation around the mean number of living offspring produced per parent animal in the control was ≤ 25 %.
- The length of the survived parent animal was measured one by one at the end of exposure.

TEST CONCENTRATIONS
- Preliminary Test (Non-GLP): To assist the selection of the test concentrations and exposure procedure, preliminary test (study No.: G18211001P) was performed prior to the definitive test. A preliminary test of acute toxicity on Daphnia magna was conducted at the WAF loading rate of 100 mg/L. After 48 hours’ exposure, no daphnia were immobilised. In order to confirm the toxic effect of test material on the reproduction of daphnia, the preliminary test of daphnia reproduction test was conducted at the WAF loading rate of 10 mg/L. Compared to the blank control group, the test material had no significant effect on the reproductive output of daphnia after 15 days’ exposure. In the preparation of the solution, the solution was turbid due to high stirring speed, it was necessary to reduce the stirring speed (the vortex height maintained at 10 %-15 % of the liquid depth in the test vessel). Since the test material is UVCB substance, the concentration of test material was analysed by LC/MS/MS and the TOC concentration of the solution was determined as auxiliary analysis meanwhile. The test material had no toxic effect on the reproduction of daphnia at nominal loading rate of 10 mg/L. The limit test could be conducted at the nominal loading rate of 10 mg/L in definitive test. As the test material is UVCB substance, the semi-static exposure was used and the solution renewed every 24 hours.
- Based on the result of the preliminary test, the definitive test was conducted as a limit test at a WAF loading rate of 10 mg/L. A blank control was run in parallel.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
reproduction
Details on results:
WATER QUALITY MEASUREMENTS
- During the period of the exposure, the temperature of the test water was maintained within the range 21.2-21.7 °C, the dissolved oxygen was in the range of 8.02-8.75 mg/L. The pH value of test solution was 7.77-8.71, the change of pH was 0.94 (not more than 1.5) units, the hardness was 160 mg/L (calculated as CaCO3). The TOC of the test water was 1.41 mg/L, which was less than 2 mg/L. The light intensity was in the range of 1040-1360 lux during the exposure period.

CONCENTRATION ANALYSIS
- LC/MS/MS analysis result: The test material was not detected in the blank control. The concentration of test material in the solutions was not maintained within the range 80 %-120 % of measured initial concentration. The geometric mean of measured concentration of test material was 0.2 mg/L for the nominal concentration of 10 mg/L.
- TOC analysis: The TOC concentration of test material corrected by the blank control was in the range of 0.31-0.84 mg/L.
- As the test material is UVCB in nature, the dissolved portion may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

BIOLOGICAL OBSERVATION
- During the test, one parent daphnia in test material treatment group was inadvertently dead and all parent daphnia in the blank control group survived.
- The time to production of the first eggs of parent daphnia in blank control and test material treatment groups was at 5-7 days of exposure and the time to the production of the first brood was at 8-9 days of exposure.
- Aborted broods: The total number of aborted broods in blank control and test material treatment groups was 22 and 19 respectively at the end of test.
- Living offspring: The number of living offspring produced per survived parent animals in blank control and test material treatment group was 140 ± 13 and 130 ± 15 (mean ± SD) respectively at the end of test. The coefficient of variation around the mean number of living offspring produced per parent animal in the control was 9 %, which was less than 25 %. Compared to the blank control group, the test material had no significant effect on the reproductive output of daphnia at the loading rate of 10 mg/L.
- The length of parent daphnia in blank control and test material treatment group was 3.93 ± 0.08 and 3.83 ± 0.08 mm (mean ± SD), respectively at the end of test.

VALIDITY OF THE TEST
- At the end of the test, the mortality of the parent animals in blank control group was 0 % (did not exceed 20 %).
- The mean number of living offspring produced per parent animal surviving in blank control at the end of the test was 140 (≥ 60).
- The accidental and inadvertent parental mortality for test material treatment group was 10 % (did not exceed 20 %).
The test satisfied all the criteria of test validity.
Reported statistics and error estimates:
The effect of the test material on the reproductive output of Daphnia magna was assessed according to the number of living offspring produced per parental animal surviving. The F test and T test was used to calculate the NOELR.

Table 1: Time to the Production of First Eggs

Loading Rate (mg/L)

Time to the Production of First Eggs (Day)

Replicates

1

2

3

4

5

6

7

8

9

10

Blank Control

6

6

6

6

6

7

6

7

6

5

10

6

6

5

6

5

6

7

6

6

6

 

Table 2: Time to the Production of First Brood

Loading Rate (mg/L)

Time to the Production of First Brood (Day)

Replicates

1

2

3

4

5

6

7

8

9

10

Blank Control

9

9

9

8

9

9

9

9

9

8

10

9

9

8

8

8

9

9

9

9

8

 

Table 3: Number of Aborted Offspring

Loading Rate (mg/L)

Number of Aborted Offspring

Total Number

Mean

SD

F-test P Value

T-test P Value

Replicates

1

2

3

4

5

6

7

8

9

10

Blank Control

0

3

2

3

2

5

3

0

2

2

22

2.2

1.5

N/A

N/A

10

/

1

3

3

3

5

0

2

0

2

19

2.1

1.6

0.79

0.45

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, on WAF loading rates, the NOELR of test material to the 21 days reproductive output of daphnia was 10 mg/L and the EL10 was > 10mg/L.
Executive summary:

The long term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 211 and other Chinese guidelines, under GLP conditions.

The objective of the study was to assess the effect of the test material on the reproductive output of Daphnia magna under semi-static conditions (test solutions were renewed every day). Based on preliminary tests, the definitive study was conducted as ‘limit’ test at 10 mg/L WAF loading. The WAF loading rate of test material of 10 mg/L and blank control were set in parallel. Ten replicates were used each group and each replicate contained one daphnid. The test duration was 21 days. The mortality of the parent animals was observed daily from the beginning of exposure. The time to production of first eggs and first brood was recorded from the fourth day of the exposure. The offspring produced by parent animal were removed daily after the production of first brood. The living offspring and aborted broods were counted. The length of the parent animal were measured one by one at the end of exposure. The NOELR and EL10 of test material to the reproductive output of daphnia were calculated.

During the test, the concentration of test material in test solutions was analysed by LC/MS/MS and TOC analyser at the beginning of exposure (0 hour), 1, 2, 8, 14 and 20 days of exposure. The test material was not detected in the blank control group, the concentration of test material in the solutions was not maintained within the range 80 %-120 % of measured initial concentration. The geometric mean of measured concentration of test material was 0.2 mg/L for the nominal concentration of 10 mg/L. The TOC concentration of test material corrected by the blank control was in the range of 0.31-0.84 mg/L. As the test material is UVCB in nature, the dissolved portion may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Compared to the blank control group, the test material had no significant effect on the reproductive output of daphnia at the loading rate of 10 mg/L. Therefore, based on the loading rate, the NOELR of test material to the reproductive output of daphnia was 10 mg/L and the EL10 was > 10mg/L.

Under the conditions of this study, on WAF loading rates, the NOELR of test material to the 21 days reproductive output of daphnia was 10 mg/L and the EL10 was > 10mg/L.

Description of key information

Under the conditions of this study, on WAF loading rates, the NOELR of test material to the 21 days reproductive output of daphnia was 10 mg/L and the EL10 was > 10mg/L.

Key value for chemical safety assessment

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

Additional information

The long term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 211 and other Chinese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to assess the effect of the test material on the reproductive output of Daphnia magna under semi-static conditions (test solutions were renewed every day). Based on preliminary tests, the definitive study was conducted as ‘limit’ test at 10 mg/L WAF loading. The WAF loading rate of test material of 10 mg/L and blank control were set in parallel. Ten replicates were used each group and each replicate contained one daphnid. The test duration was 21 days. The mortality of the parent animals was observed daily from the beginning of exposure. The time to production of first eggs and first brood was recorded from the fourth day of the exposure. The offspring produced by parent animal were removed daily after the production of first brood. The living offspring and aborted broods were counted. The length of the parent animal were measured one by one at the end of exposure. The NOELR and EL10 of test material to the reproductive output of daphnia were calculated.

During the test, the concentration of test material in test solutions was analysed by LC/MS/MS and TOC analyser at the beginning of exposure (0 hour), 1, 2, 8, 14 and 20 days of exposure. The test material was not detected in the blank control group, the concentration of test material in the solutions was not maintained within the range 80 %-120 % of measured initial concentration. The geometric mean of measured concentration of test material was 0.2 mg/L for the nominal concentration of 10 mg/L. The TOC concentration of test material corrected by the blank control was in the range of 0.31-0.84 mg/L. As the test material is UVCB in nature, the dissolved portion may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Compared to the blank control group, the test material had no significant effect on the reproductive output of daphnia at the loading rate of 10 mg/L. Therefore, based on the loading rate, the NOELR of test material to the reproductive output of daphnia was 10 mg/L and the EL10 was > 10mg/L.

Under the conditions of this study, on WAF loading rates, the NOELR of test material to the 21 days reproductive output of daphnia was 10 mg/L and the EL10 was > 10mg/L.