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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:
2017-03-20 - 2017-04-13
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:
The deviations were considered to have not affected the integrity or validity of the study.
Deviations:
yes
Remarks:
No lux reading in the range-finding test at 24h. No analysis of the range finding samples. Total Organic Carbon (TOC) analysis was carried out on the samples in the definitive test.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
The deviations were considered to have not affected the integrity or validity of the study.
Deviations:
yes
Remarks:
No lux reading in the range-finding test at 24h. No analysis of the range finding samples. Total Organic Carbon (TOC) analysis was carried out on the samples in the definitive test.
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of The United Kingdom
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The test samples were thawed with the aid of a water bath, filtered through a 0.45 μm cellulose acetate filter, and then analyzed.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the General Physico-Chemical Properties of the test item indicated that the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. A 23-Hour stirring period, followed by a 1-Hour standing period was deemed sufficient to ensure equilibration between the test item and aqueous phase.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: derived from in-house laboratory cultures.
- Age of parental stock (mean and range, SD): less than 24 hours old.
- Feeding during test: no food during exposure


ACCLIMATION
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortalities observed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period described.
Hardness:
not reported
Test temperature:
21 - 22 °C
pH:
7.5 - 7.6
Dissolved oxygen:
8.5 - 8.6 mg O2/L
Salinity:
not applicable
Conductivity:
not reported
Nominal and measured concentrations:
range-finding test: 5.0 and 50 mg/L loading rate WAF
definite test: 50 mg/L loading rate WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass jars
- Type (delete if not applicable): closed (covered)
- Material, size, headspace, fill volume: glass, filled with 100 mL of test preparation
- Aeration: none
- No. of organisms per vessel: 5 (definitive test); 5 (range-finding test)
- No. of vessels per concentration (replicates): 4 (definitive test); 1 (range-finding test)
- No. of vessels per control (replicates): 4 (definitive test); 1 (range-finding test)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M7)
- Culture medium different from test medium: no
- Intervals of water quality measurement: measurements at 0 (fresh media) and 48 hours (old media). The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: between 200 and 1200 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Immobilisation was recorded daily (24 h and 48 h after start of exposure). Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 5.0 and 50 mg/L loading rate WAF
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Immobility of test organisms was not observed.
- Mortality of control: no (A sub-lethal effect of exposure was observed in the mineral oil control group. This response was a single daphnid trapped at the surface)
- Other adverse effects control: no
- Abnormal responses: none
Results with reference substance (positive control):
- Valid reference substance used
- EC50/LC50: 1.2 mg/L (1.1 - 1.3 mg/L 95% Confidence Limits) after 48 hours
- Other: The results from the positive control with potassium dichromate were within the normal range for this reference material.
Reported statistics and error estimates:
An estimate of the EL50 values was given by inspection of the immobilization data.

Range-finding Test

No immobilization was observed at 5.0 and 50 mg/L loading rate WAF. A single daphnid was observed to be immobilized in the mineral oil control group after 48 hours exposure, however, this was considered to be due to natural causes given that less than 10% effect was observed.

Based on this information, a single loading rate of four replicates, of 50 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Table 1 Cumulative Immobilization Data and Observations in the Range-finding Tests

Nominal Loading Rate (mg/L) 24 Hours
Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate) Observations
R1 R2 R3 R4 Total % R1 R2 R3 R4
Control 0 0 0 0 0 0 5N 5N 5N 5N
Mineral Oil Control 0 0 0 0 0 0 5N 5N 1T4N 5N
5.0 0 0 0 0 0 0 5N 1T4N 5N 5N
50 0 0 0 0 0 0 5N 5N 5N 5N
Nominal Loading Rate (mg/L) 48 Hours
Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate) Observations
R1 R2 R3 R4 Total % R1 R2 R3 R4
Control 0 0 0 0 0 0 6N* 5N 5N 5N
Mineral Oil Control 0 0 1 0 1 5 5 N 5N 4N 5N
5.0 0 0 0 0 0 0 5N 4N1T 5N 5N
50 0 0 0 0 0 0 5N 5N 5N 5N

* 6 daphnids observed, no impact on outcome of test

N = No sub-lethal effects observed

T =Trapped at surface

R1 – R4 = Replicates 1 to 4

Definitive Test

Chemical Analysis of Test Loading Rates

Chemical analysis of the fresh test preparation at 0 hours showed that a measured concentration of 0.14 mg/L as boron (equivalent to 3.74 mg/L test item) was obtained. Chemical analysis of the aged test preparation at 48 hours showed that a measured concentration of 0.15 mg/L as boron (equivalent to 3.91 mg/L test item) was obtained. Analysis of the control and mineral oil control at 0 and 48 hours showed that measured concentrations of less that the Limit of Quantification (LOQ) of the analytical method employed, determined to be 0.050 mg/L as boron, were obtained.

Total Organic Carbon Analysis

Samples of the control, mineral oil control and the 50 mg/L loading rate WAF were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.

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.

Immobilization Data

There was no immobilization in 20 daphnids exposed to a 50 mg/L loading rate WAF for a period of 48 hours.

The No Observed Effect Loading rate after 24 and 48 hours exposure was 50 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L.

Table 2 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
R1 R2 R3 R4 Total % R1 R2 R3 R4
Control 0 0 0 0 0 0 5N 5N 5N 5N
Mineral Oil Control 0 0 0 0 0 0 5N 5N 1T4N 5N
50 0 0 0 0 0 0 5N 5N 5N 5N
Nominal Loading Rate (mg/L) 48 Hours
Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate) Observations
R1 R2 R3 R4 Total % R1 R2 R3 R4
Control 0 0 0 0 0 0 5N 5N 5N 5N
Mineral Oil Control 0 0 0 0 0 0 5 N 5N 1T4N 5N
50 0 0 0 0 0 0 5N 5N 5N 5N

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

T = Trapped at surface

Water Quality Criteria

Temperature was maintained at 21 °C to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Throughout the test the light intensity was observed to be in the range 476 to 482 lux.

Table 3 Water Quality Measurements

Nominal Loading Rate (mg/L) 0 Hours 24 Hours 48 Hours
pH mg O2/L T °C T °C pH mg O2/L T °C
Control R1 7.5 8.5 22 22 7.5 8.6 21
Mineral Oil Control R1 7.6 8.5 22 22 7.5 8.6 21
50 R1 7.6 8.5 22 22 7.5 8.6 21

R1 = Replicate 1

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

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 mineral oil control was observed to be a clear colorless water column with colourless oily globules on the surface whilst the 50 mg/L loading rate WAF was observed to be a clear colourless water column with amber colored oily globules on the surface. After 23 hours stirring and a 1-Hour standing period the mineral oil control was observed to be a clear colorless water column with colorless oily globules of test item on the surface whilst the 50 mg/L loading rate was observed to be a clear colorless water column with a waxy white layer dispersed with amber globules on the surface and a small amount/round ball of test item on the bottom. 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, mineral oil control and test solutions were observed to be clear colorless solutions.

Positive Control

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

Time Point (Hours) EC50(mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 1.3 1.0 - 1.8 1.0 1.8
48 1.2 1.1 - 1.3 0.56 1.0

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item*.

* The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give an EC50-24 hour for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading rate was 50 mg/L loading rate WAF.
Executive summary:

The acute toxicity of the test substance towards Daphnia magna was determined according to OECD Guideline 202 and Method C.2 of Commission Directive 92/69/EEC in compliance with GLP. Given a low water solubility of the test item of less than 1.0 mg C/L it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 50 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The test item was a complex mixture containing 53% mineral oil. At the Sponsors request an additional vessel was prepared containing mineral oil at the same concentration as in the test item preparations; 26.5 mg/L.

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

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 EL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at concentrations in excess of 50 mg/L.

Description of key information

Daphnia sp., 48-Hour Acute Immobilization Test, OECD 202, static, fresh water: EL50 (48h) > 50mg/L loading rate WAF, NOEL (48h) 50 mg/L loading rate WAF based on immobilization

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
50 mg/L

Additional information

In the key study (Ablitt, 2017c) the acute toxicity of the test substance towards Daphnia magna was determined according to OECD Guideline 202 and Method C.2 of Commission Directive 92/69/EEC in compliance with GLP. Given a low water solubility of the test item of less than 1.0 mg C/L it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 50 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The test item was a complex mixture containing 53% mineral oil. At the Sponsors request an additional vessel was prepared containing mineral oil at the same concentration as in the test item preparations; 26.5 mg/L.

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

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 EL50values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at concentrations in excess of 50 mg/L.