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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2018 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 0.10, 0.32, 1.0, 3.2, 10 mg/L
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
Nominal amounts of test item (5.0, 16 and 50 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 3.2 and 10 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L test concentration to give further test concentrations of 0.10 and 0.32 mg/L loading rate WAF.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5, originally supplied by University of Sheffield/UK
- Source: in-house laboratory cultures
- Feeding during test: no

ACCLIMATION
- Acclimation conditions: Elendt M7 medium
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
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:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 °C to 22 ºC
pH:
7.9 - 8.0 at test start; 7.7 - 7.8 at end of exposure period
Dissolved oxygen:
8.6 - 8.8 mg/L at test start; 8.7 - 8.9 mg/L at end of exposure period
Nominal and measured concentrations:
nominal loading rates: Control, 0.10, 0.32, 1.0, 3.2, 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL beakers containing approximately 100 mL of test preparation
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h darkness with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure

RANGE-FINDING STUDY
- Test concentrations: Control, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study:
Immobilization was observed at all test concentrations. Based on the results of the range finding test loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test. Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 8.6 to 165 mg/L. Chemical analysis of the test preparations at 48 hours showed measured test concentrations of 5.2 to 179 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
8.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: No sub lethal effects of exposure were observed throughout the test at test concentrations of 0.10, 0.32, 1.0 and 3.2 mg/L loading rate WAF. A sub lethal effect of exposure was observed 10 mg/L loading rate WAF. The response was reduced mobility.
- Mortality of control: 10% mortality in control (2/20)

- Observations on Test Item Solubility:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 1.0, 3.2 and 10 mg/L test concentrations were observed to be clear colorless water columns with test item floating on the surface. After 23 hours stirring and a 1 Hour standing period the test concentrations were observed to remain as at the start of mixing. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present.
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 24 h EC50 = 0.79 mg/L (95% c.i. 0.73 - 0.86 mg/L)
- The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24 Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L
Reported statistics and error estimates:
The EL50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum Likelihood regression. The Lowest Observed Effect Loading rate (LOEL) and the No Ovserved Effect Loading Rate (NOEL) rate at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Loading Rate
(mg/L)

Observations (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Control

0

0

5 N

5 N

0

0

5 N

5 N

1.0

1

0

4 N

5 N

1

0

4 N

5 N

10

5

5

AI

AI

5

5

AI

AI

100

5

5

AI

AI

5

5

AI

AI

 

 

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

1

1

0

2

10

5 N

4 N

4 N

5 N

0.10

0

1

0

0

1

5

5 N

4 N

5 N

5 N

0.32

0

0

0

1

1

5

5 N

5 N

5 N

4 N

1.0

2

0

1

0

3

15

3 N

5 N

4 N

5 N

3.2

0

0

1

0

1

5

5 N

5 N

4 N

5 N

10

2

3

1

5

11

55

3 N

2 R

4 N

AI

 

Nominal Loading Rate
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

1

1

0

2

10

5 N

4 N

4 N

5 N

0.10

0

2

0

0

2

10

5 N

3 N

5 N

5 N

0.32

0

1

0

1

2

10

5 N

4 N

5 N

4 N

1.0

2

1

1

0

4

20

3 N

4 N

4 N

5 N

3.2

0

0

1

0

1

5

5 N

5 N

4 N

5 N

10

2

5

2

5

14

70

3 N

AI

3 N

AI

 

Rep= Replicate

N = Normal

R = Reduced mobility

AI = All daphnia immobilized

Validity criteria fulfilled:
yes
Conclusions:
The 48 h EL50 of CGE-PMDA adduct was 8.4 mg/L (nominal loading rate WAF).
Executive summary:

A study was performed to assess the acute toxicity of CGE-PMDA adduct to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary rangefinding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of0.10,0.32,1.0,3.2and10 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.10 to 5.3 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.076 to 5.8 mg/L.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure of Daphnia magna to the test item gave the following results:

 

 

Time Point
(Hours)

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

NOEL
(mg/L)

LOEL
(mg/L)

48

8.4

Not determined due to the nature of the data

3.2

10

 

Description of key information

48 h EL50 = 8.4 mg/L (OECD TG 202; Daphnia magna; WAF)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
8.4 mg/L

Additional information

A study was performed to assess the acute toxicity of CGE-PMDA adduct to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary rangefinding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10,0.32,1.0,3.2 and 10 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.10 to 5.3 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.076 to 5.8 mg/L.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure of Daphnia magna to the test item gave the following results:

 

 

Time Point
(Hours)

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

NOEL
(mg/L)

LOEL
(mg/L)

48

8.4

Not determined due to the nature of the data

3.2

10