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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:
Dec 2013 - Feb. 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP OECD guideline study with analytical monitoring, well described
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 3, 6, 10, 13, 17 and 20 and of the expired test preparations on Days 1, 4, 7, 11, 14, 18 and 21. Samples taken on Day 1 were stored frozen prior to analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary. All sample bottles were shielded from light.
Details on test solutions:
An amount of test item (500 mg) was dissolved in test water and the volume adjusted to 5 liters to give the 100 mg/L test concentration. Aliquots (20, 64, 200 and 640 mL) of the 100 mg/L test concentration were separately added to a final volume of 2 liters of test water to give the remaining test concentrations of 1.0, 3.2, 10 and 32 mg/L respectively. Due to the light sensitive nature of the test item, all preparation was carried out under non actinic light.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 3, 6, 10, 13, 17 and 20 (fresh media) and on Days 1, 4, 7, 11, 14, 18 and 21 (old media).
Test organisms (species):
Daphnia magna
Details on test organisms:
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 deg.C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
190 to 225 mg/L as CaCO3
Test temperature:
20 deg. C
pH:
7.6 - 8.0
Dissolved oxygen:
7.9 - mg O2/L
Nominal and measured concentrations:
Nominal (mg/L): 1.0; 3.2; 10; 32; 100
Mean Measured (mg/L): 0; 0; 2.0; 7.2; 26
Details on test conditions:
3.4.2.2 Exposure Conditions
For each concentration a single daphnid was placed in approximately 160 mL of the test preparation in completely filled and sealed 160 mL glass vessels. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at approximately 20 C in darkness for 21 days. Each vessel was randomly assigned to a position in the laboratory. The test vessels were not aerated. The diluent water only was aerated prior to use.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were renewed daily. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.

Each daphnid received approximately 2 to 7 µL of an algal suspension (Desmodesmus subspicatus) and approximately 16 to 55 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
parental Daphnia generation (P1)
Remarks on result:
other: 10 - 15 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 7.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
7.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Reported statistics and error estimates:
The EC50 (immobilization) values and associated confidence limits on Day 21 were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).

The EC50 (reproduction) value after 21 days was estimated by inspection of the data

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

The observed mortalities in the parental (P1) generation of the 10 and 32 mg/L test groups were compared to the control group using the corrected chi-squared statistic (Breslow and Day 1980).

For the estimation of the "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) the numbers of live young produced per adult over the duration of the test for the control the 1.0, 3.2, 10 and 32 mg/L test groups were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and the Williams test for differences between treatment means when several dose levels are compared with a zero dose control (Williams 1971) (see Appendix 4). Results from the control and the 1.0, 3.2, 10 and 32 mg/L test groups Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 4). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

The 100 mg/L test group data was not included in the statistical analysis as the effects of exposure eliminated all the daphnids prior to termination of the test.

Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Nominal

Concentration

(mg/L)

%

Survival

of P1

Number of

Live Young*

  

Number of

Dead Young

  

Number of

Unhatched Eggs

  

 

Per Female

(cumulative)

 

 

Per Female

(cumulative)

 

 

Per Female

(cumulative)

 

Total

Total

Total

 

 

 

Control

100

625

63

0

0

0

0

1.0

100

608

61

0

0

0

0

3.2

100

609

61

0

0

0

0

10

80

515

62

0

0

0

0

32

90

563

63

0

0

0

0

100

0

19

2

0

0

0

0


*The number of live young per live adult at Day 21

Nominal Concentration (mg/L)

Day

20+

21*

Control

mg O2/L

9.1

7.1

pH

7.6

7.4

T°C

20

21

1.0

mg O2/L

9.1

7.2

pH

7.6

7.3

T°C

20

21

3.2

mg O2/L

9.0

7.5

pH

7.5

7.3

T°C

20

21

10

mg O2/L

9.2

8.0

pH

7.5

7.3

T°C

20

21

32

mg O2/L

9.0

7.9

pH

7.5

7.4

T°C

20

21

100

mg O2/L

A/D

pH

T°C


+After renewal

*Before renewal

A/DAll Dead

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the experiment, the 21 d NOEC is 7.2 mg/L
Executive summary:

SUMMARY

Introduction

A study was performed to assess the toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "DaphniamagnaReproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

 

 

Methods…….

Based on the results of a preliminary range-finding test,Daphnia magnawere exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The test solutions were renewed daily. 

 

The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin®flake food suspension.

 

 

Results…….

Analysis of the 10, 32 and 100 mg/L test preparations showed measured test concentrations to range from 12% to 43% of nominal value and so the results are based mean measured test concentrations. Low measured concentrations were obtained at the nominal concentrations of 10, 32 and 100 mg/L and this was considered to be due to the highly volatile nature of the test item. Variable measured concentrations were also obtained for the 1.0, 3.2 and 10 mg/L test preparations, however, as no effects were observed at these test concentrations, and these were not required for any of the statistical analysis, this was considered not to have an impact on the test. 

 

Exposure of Daphnia magna to the test item gave the following results based on the mean measured test concentrations:

   

Endpoint

Concentration (mg/L)

Immobilization

EC10

-

 

95% confidence limits

-

 

EC50

12

 

95% confidence limits

10 - 15

Reproduction

EC10

-

 

95% confidence limits

-

 

EC50

>7.2

 

95% confidence limits

-

No Observed Effect Concentration (NOEC)

7.2

Lowest Observed Effect Concentration (LOEC)

26

Maximum Acceptable Toxicant Concentration (MATC)

14

 

Description of key information

One OECD 211 GLP study is available with a 21 d NOEC of 7.2 mg/L

Key value for chemical safety assessment

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

Additional information

Based on the results of a preliminary range-finding test,Daphnia magnawere exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The test solutions were renewed daily. 

 

The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin®flake food suspension.

Analysis of the 10, 32 and 100 mg/L test preparations showed measured test concentrations to range from 12% to 43% of nominal value and so the results are based mean measured test concentrations. Low measured concentrations were obtained at the nominal concentrations of 10, 32 and 100 mg/L and this was considered to be due to the highly volatile nature of the test item. Variable measured concentrations were also obtained for the 1.0, 3.2 and 10 mg/L test preparations, however, as no effects were observed at these test concentrations, and these were not required for any of the statistical analysis, this was considered not to have an impact on the test.