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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:
10 November 2017 to 14 September 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Physical state/Appearance: Brown viscous liquid
Purity: >90%
Analytical monitoring:
yes
Details on sampling:
Samples from the control and the 100 mg/L loading rate WAF test group were taken from the freshly prepared bulk test preparation at 0 and 24 hours and from the old pooled replicates at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis. Analysis was not performed at the concentrations below the NOEL (10 and 18 mg/L loading rates).
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and the complex nature of the test item, the test medium was prepared as Water Accommodating Fraction ((WAF) based on a the methods established in a preliminary mixing trial.

Nominal amounts of test item were each separately added to the surface of test water to achieve nominal loading rates of 10, 18, 32, 56 and 100 mg/L. 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 10, 18, 32, 56 and 100 mg/L loading rate WAFs.

Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory culture
- Culture medium (dilution water): Elendt M7 medium
- Culture conditions: 18 to 22 °C; 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Age: Less than 24 hours old
- Food: Culture was fed with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Frequency: Daily

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:
other: Reconstituted water (ISO medium)
Limit test:
yes
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
21 to 22 °C
pH:
7.6 to 7.9
Dissolved oxygen:
8.3 to 9.0 mg O2/L
Nominal and measured concentrations:
Nominal loading rates (WAF) were 0 (control), 10, 18, 32, 56 and 100 mg/L.

Chemical analysis of the 32, 56 and 100 mg/L loading rate fresh test preparations at 0 and 24 hours showed measured test concentrations ranging from 0.21 to 0.63 mg/L as molybdenum. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.23 to 0.61 mg/L as molybdenum.

The dissolved test item may have been comprised of one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, the results were based on nominal loading rates only.
Details on test conditions:
TEST SYSTEM
Test solutions were prepared in the same manner as described for the range-finding test. Glass jars (150 mL) containing approximately 100 mL of test solution were used. At the start of the test five daphnids were placed in each test and control vessel at random, containing 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. Throughout the test, the light intensity ranged from 837 to 927 lux.

The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test item. Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 Hour old test media into the fresh test media.

OTHER TEST CONDITIONS
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 Hour old test preparations. The appearance of the test media was recorded daily.

RANGE-FINDING TEST
Daphnia magna were exposed to a series of nominal loading rates (WAF) of 1.0, 10 and 100 mg/L. Five daphnids each were placed in duplicate test and control vessels and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of +/-1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. The control group was maintained concurrently under identical conditions, but was not exposed to the test item. Semi-static test conditions were employed in the range finding test in an effort to maintain dissolved test item concentrations. For the test mediium renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 hour old test media into the fresh test media. A sample of each loading rate WAF was taken for chemical analysis at 0 and 24 hours in order to assess the stability of the test solutions under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
Loading Rate (WAF)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
loading rate WAF
Basis for effect:
mobility
Remarks on result:
other: (95% Confidence Limits: 66 - 120 mg/L loading rate WAF)
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
loading rate WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ Loading Rate (WAF)
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
No immobilization was observed at 10 mg/L loading rate WAF, however, immobilization was observed at 1.0 (20%) and 100 mg/L loading rate WAF (90%). A sub lethal effect of exposure (reduced mobility) was observed in one daphnia at 100 mg/L loading rate WAF. Based on this information, loading rates of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

DEFINITIVE TEST
No sub lethal effects of exposure were observed throughout the test at concentrations of 10 and 18 mg/L loading rate WAF. Reduced mobility was observed at 56 and 100 mg/L loading rate WAF.
Results with reference substance (positive control):
A positive control (Envigo study number LK67NP), using potassium dichromate as the reference item, was conducted on January 23 -25, 2018, 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. The 48-hour EC50 was 0.75 mg/L (95% Confidence Limits: 0.56 - 1.0 mg/L) and the NOEC was 0.56 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
The loading rate causing 50% immobilization in the treatment group (EL50) at 24 hours was estimated by visual inspection of the data. The EL50 value and associated confidence limits at 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 (LOEL) rate and the NOEL rate at 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Cumulative Immobilization Data and Observations in the Definitive Test

 

Cumulative Immobilization
(5 Daphnia/Rep at Test Initiation)

 

 

 

 

 

Observations

 

 

 

Nominal Loading Rate
(mg/L)

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

24 hours

 

 

 

 

 

 

 

 

 

 

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

48 hours

 

 

 

 

 

 

 

 

 

 

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

1

1

5

5 N

5 N

5 N

4 N

56

2

3

1

0

6

30

3 R

2 R

4 R

5 R

100

4

3

1

4

12

60

1 R

2 R

4 R

1 R

Rep = Replicate

N = Normal

R = Reduced mobility

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item for 48 hours resulted in an EL50 value of 82 mg/L loading rate WAF. The No Observed Effect Loading Rate was 32 mg/L loading rate WAF.
Executive summary:

A study was conducted in accordance with OECD Chemicals Testing Guideline No. 202 and EU Test Method C.2, under GLP conditions, to assess the acute toxicity of the test item to the freshwater invertebrate, Daphnia magna. 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 range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item at nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of approximately 21°C to 22°C under semi-static test conditions. The number of immobilized daphnids and any adverse reactions to exposure were recorded after 24 and 48 hours. 

Chemical analysis of the freshly prepared 32, 56 and 100 mg/L loading rate WAF solutions collected at 0 and 24 hours showed measured test concentrations ranging from 0.21 to 0.63 mg/L as molybdenum. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.23 to 0.61 mg/L as molybdenum. Analysis was not performed at the concentrations below the NOEL (10 and 18 mg/L loading rates). 

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

Exposure of Daphnia magna to the test item for 48 hours resulted in EL50 value of 82 mg/L loading rate WAF and the No Observed Effect Loading Rate (NOEL) was 32 mg/L loading rate WAF.

Description of key information

A study was conducted according to OECD Guideline 202 and EC Method C.2, under GLP, to assess the acute toxicity of the test item to the freshwater invertebrate, Daphnia magna. Exposure of Daphnia magna to the test item for 48 hours resulted in EL50 value of 82 mg/L loading rate WAF and the No Observed Effect Loading Rate was 32 mg/L loading rate WAF (Envigo, 2018).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
82 mg/L

Additional information