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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:
August 2013 - January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 3706OB
- Expiration date of the lot/batch: 31 December 2018
- Purity test date: not applicable - complex mixture

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark


Analytical monitoring:
yes
Details on sampling:
Range finding test:
A sample of each loading rate WAF ( 1.0, 10 and 100 mg/L) was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive Test:
Water samples were taken from the control and each loading rate WAF (10, 18, 32, 56 and 100 mg) at 0 and 48 hours for quantitative analysis (replicates R1 - R4 pooled).
Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:
no
Details on test solutions:

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

PREPARATION AND APPLICATION OF TEST SOLUTION:
The requested amounts of test item were each separately added to the surface of test water to give the requested loading rates. 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. . Microscopic observations made on the WAFs (range finding and definitive tests) indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no microscopic particles of test item.
Due to the light-sensitive nature of the test item, all preparations were performed under non-actinic lighting conditions and the stirring vessels, test vessels and sample bottles were shielded from light.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: no data
- Source: in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water - ISO Medium had an approximate theoretical total hardness of 250 mg/L as CaC03.
Test temperature:
Temperature ranged between 20 to 20°C
pH:
pH ranged between 7.7 to 8.0
Dissolved oxygen:
Dissolved oxygen ranged between 8.5 to 8.8 mg O2/L
Salinity:
no data reported
Conductivity:
no data reported
Nominal and measured concentrations:
Range finding test:
1.0, 10 and 100 mg/L loading rates (WAF)

Definitive test:
1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates (WAF)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type : closed
- Renewal rate of test solution: none, static
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

EXPOSURE CONDITION
In the definitive test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 21 °C. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The test was carried out in darkness due to the light-sensitive nature of the test item.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period

TEST MEDIUM
- Source: Reconstituted water (ISO medium) as recommanded in the OECD 202 was used for both the range-finding and definitive tests

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: none, The test was carried out in darkness due to the light-sensitive nature of the test item.

EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure
- Water Quality Criteria: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L loading rates (WAF)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate .
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: WAF Water Accommondated Fraction
Remarks:
results based on nominal loading rate
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: WAF WAter Accommondated Fraction
Remarks:
Results based on nominal loading rate
Details on results:
RANGE-FINDING TEST:
No immobilization was observed at 1.0 mg/L loading rate WAF. However, immobilization was observed at 10 and 100 mg/L loading rate WAF. (Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 in "any other information on results including table").
Analysis of the 1.0, 10 and 100 mg/L loading rate WAFs at 0 hours showed measured concentrations of 0.0015, 0.022 and 0.071 mg/L respectively. Analysis of the 1.0, 10 and 100 mg/L loading rate WAFs at 48 hours showed measured concentrations 0.00052, 0.022 and 0.066 mg/L respectively. The slight decline in measured concentration observed in the 1.0 mg/L loading rate over the test period was due to an error in sampling, whereby the control was sampled in error and not discarded prior to sampling the 1.0 mg/L loading rate, rather than true instability of the test item.

DEFINITIVE TEST:
- Immobilization Data:
The EL50 were 9.5 and 8.0 mg/L loading rate WAF after 24 and 48 hours exposure respectively.
The No Observed Effect Loading rate after 24 and 48 hours exposure was 5.6 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 10 mg/L loading rate WAF. (Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 in "any other information on results including table").
Significant immobilization was observed in the 1.8 mg/L loading rate WAF test group. However, this was considered not to be a true toxic effect of the test item as this did not follow a concentration - dependent pattern. In addition, both surviving and immobilized daphnids in this test group were observed to be trapped on the surface. As this effect was not observed in any other test group, this effect was considered not to be a true toxic effect of the test item but due to possible contamination of the test preparations. Single immobilized daphnids were observed in the control and 3.2 mg/L loading rate WAF groups however these were considered not to be true toxic effects as less than 10% immobilization occurred.

- Chemical Analysis of Test Loading Rates:
Analysis of the 5.6 and 10 mg/L loading rate WAFs at 0 hours showed measured concentrations of 0.0086 and 0.016 mg/L respectively. Analysis of the 5.6 and 10 mg/L loading rate WAFs at 48 hours showed measured concentrations of 0.0081 and 0.016 mg/L respectively.
The toxicity of the test item cannot be attributed to a single component or a mixture of components but to the test item as a whole. In addition, the measured concentrations obtained for the WAFs are not stability indicating and are rather a confirmation that the test item is present. Given these factors the results of the study were based on nominal loading rates only.

OBSERVATION ON THE 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, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed tobe clear colorless water columns with particles of test item floating on the surface and suspended in the water column. After 23 hours stirring the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates wereobserved to be clear colorless water columns with particles of test item floating on the surface, suspended in the water column and settled at the base of the vessel. After a 1-Hour standing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear colorless water columns with particles of test item floating on the surface and settled at the base of the vessel. The WAFs were then removed by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed that the glass wool plug had removed all of the microscopic particles of test item from the media. During the test the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear, colorless solutions.
Results with reference substance (positive control):
- Results with reference substance valid: yes, A positive control (Harlan Study Number 41301833) used potassium dichromate as the reference item
- EC50: EC50 (24h) = 1.0 mg/L (95% Confidence Limits 0.91-1.2) / EC50 (48h) = 0.71 (95% Confidence Limits 0.65 - 0.76)
The results from the positive control with potassium dichromate were within the normal range for this reference item. The positive control was conducted between 16 May 2013 and 18 May 2013
Reported statistics and error estimates:
The EL50 values and associated confidence limits at 24 and 48 hours were calculated by the Trimmed-Spearman method (Hamilton et al9 1977) using the ToxCalc computer software package (ToxCalc, 1999).

Table 1 : Cumulative Immobilization Data in the Range-finding Test

Nominal Loading Rate (mg/L)

Cumulative Immobilized Daphnia, (Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

1.0

0

0

10

10

10

100

10

10

Table 2:   Cumulative Immobilization Data in the Definitive Test

Nominal

Loading Rate

(mg/L)

 

 

 

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

1

0

0

1

5*

0

l

0

0

1

5*

1.0

0

0

0

0

0

0

0

0

0

0

0

0

1.8

1

0

0

5

6

30**

1

0

1

5

7

35**

3.2

0

0

0

1

1

5*

0

0

0

1

1

5*

5.6

0

0

0

0

0

0

0

0

0

0

0

0

10

2

4

1

4

11

55

5

5

2

4

16

80

*Not considered to be a true toxic effect as less than 10% immobilization observed.

** Immobilization observed not considered to be a true toxic effect of the test item as considered not to follow a concentration - dependent pattern

 

Analytical results:

Table 3 : Results for test samples (Range finding)

Time Point

[hours]

Nominal Loading Rate

Cnom

[mg/L]

Measured

Concentration

of Test Item in

Sample Vial

X

[mg/L]

Sample

Preparation

Factor

F

Determined

Concentration

of Test Item in

Range-Finding

Sample

c [mg/L]

0

1.0

10

100

0.0608

0.880

2.86

0.025

0.025

0.025

0.00152

0.0220

0.0714

48

1.0

10

100

0.0207

0.888

2.62

0.025

0.025

0.025

0.000517

0.0222

0.0655        

Table 4 : Results for test samples (definitive test)

Time Point      

 [hours]

NominalLoading Rate

Cnom

 

[mg/L]

Measured Concentration of Test Item in Sample Vial

X

[mg/L]

Sample Preparation Factor

F

Determined concentration of Test Item in Test Sample

c

[mg/L]

0

Control

<LOQ

0.025

<LOQ

5.6

0.343

0.025

0.00858

10

0.640

0.025

0.0160

48

Control

<LOQ

0.025

<LOQ

5.6

0.325

0.025

0.00813

10

0.636

0.025

0.0159

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave the following results:
The 48h-EL50 were 8.0 mg/L loading rate WAF (with a 95% Confidence Limits 7.4 - 8.7 (mg/L Loading Rate WAF))
The No Observed Effect Loading rate after 24 and 48 hours exposure was 5.6 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 10 mg/L loading rate WAF
Executive summary:

The effect of the test item on the mobility of species Daphnia magna was investigated in a 48hour static test according to 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. The study was compliant with the GLP.

Due to the low aqueous solubility and complex nature of the test item, the test was performed using the loading rate (according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), thus all the test solutions were prepared as a Water Accommodated Fraction (WAF). 

Based on a preliminary range-finding test, twenty daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L and control for 48 hours at a temperature of approximately 21°C under static test conditions. The 20 daphnids per test solution, divided into four replicates of 5 daphnids each, were observed for immobility after 24 and 48 hours of exposure

Analysis of the 5.6 and 10 mg/L loading rate WAFs at 0 hours showed measured concentrations of 0.0086 and 0.016 mg/L respectively. Analysis of the 5.6 and 10 mg/L loading rate WAFs at 48 hours showed measured concentrations of 0.0081 and 0.016 mg/L respectively. The toxicity of the test item cannot be attributed to a single component or a mixture of components but to the test item as a whole. In addition, the measured concentrations obtained for the WAFs are not stability indicating and are rather a confirmation that the test item is present. Given these factors the results of the study were based on nominal loading rates only.

The EL50 were 9.5 and 8.0 mg/L loading rate WAF after 24 and 48 hours exposure respectively. The No Observed Effect Loading rate after 24 and 48 hours exposure was 5.6 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 10 mg/L loading rate WAF.

The two validity criteria of the OECD guideline 202 were fulfilled, therefore this study is considered as reliable without restrictions.

Description of key information

One reliable study is available for the Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) for this endpoint. The acute immobilization (EC50 and NOEC) of the Reaction mass of AminoPhosphonium salt and Bisphenol AF towards Daphnia magna was determined in a 48hour static test according to 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. The study was compliant with the GLP.

Due to the low aqueous solubility and complex nature of the test item, the test was performed using the loading rate (according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), thus all the test solutions were prepared as a Water Accommodated Fraction (WAF). 

Based on a preliminary range-finding test, twenty daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L and control for 48 hours at a temperature of approximately 21°C under static test conditions. The 20 daphnids per test solution, divided into four replicates of 5 daphnids each, were observed for immobility after 24 and 48 hours of exposure

Analysis of the 5.6 and 10 mg/L loading rate WAFs at 0 hours showed measured concentrations of 0.0086 and 0.016 mg/L respectively. Analysis of the 5.6 and 10 mg/L loading rate WAFs at 48 hours showed measured concentrations of 0.0081 and 0.016 mg/L respectively. The toxicity of the test item cannot be attributed to a single component or a mixture of components but to the test item as a whole. In addition, the measured concentrations obtained for the WAFs are not stability indicating and are rather a confirmation that the test item is present. Given these factors the results of the study were based on nominal loading rates only.

 

The EL50 were 9.5 and 8.0 mg/L loading rate WAF after 24 and 48 hours exposure respectively. The No Observed Effect Loading rate after 24 and 48 hours exposure was 5.6 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 10 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
8 mg/L

Additional information

The Key value is expressed as loading rate (WAF) i.e. EL50 (48h) = 8 mg/L