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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:
23 February 2018 to 8 January 2019
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
CAS RN: 1412893-77-6
Purity: >94% (This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB))
Description: Brown paste to very viscous liquid
Analytical monitoring:
yes
Details on sampling:
At the start of the test (0 hours) and at media renewal at 24 hours, 10 mL samples of freshly prepared test media were taken from the control and the test media preparation vessel for chemical analysis. At 24 and 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media from the test and control groups. In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Concentrations of the test substance were determined by diluting aqueous samples with acetonitrile containing 0.2% formic acid, then diluting further with Elendt M4 medium: acetonitrile (1:1, v/v) containing 0.1% formic acid as required to bring the response within the calibration range.
Vehicle:
no
Details on test solutions:
The test material is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with variable water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guidance, the test substance was prepared as Water Accommodated Fractions (WAFs). Due to the toxicity of the test material it was necessary to prepare a single WAF at a loading rate of 1.0 mg/L followed by dilutions to prepare the remaining test loading rates.

At the start of the test and media renewal at 24 hours, ca 1.0 mg of test material was weighed onto a glass slide and placed in 1000 mL of Elendt M4 medium. The preparation was stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparation was allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 1.0 mg/L loading rate WAF. Dilutions were prepared from the 1.0 mg/L loading rate WAF to give the test series. A control group was prepared in the same manner without the addition of test material.

The test preparations were observed to be colourless solutions throughout the duration of the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Test organism: Juvenile Daphnia magna (Straus), less than 24 hours old
- Source: Parental laboratory cultures, held and maintained at the test facility (culture was initiated from ephippia obtained from MicroBio Tests Inc., Belgium)
- Age of parental stock: Less than 24 hours old
- Food type: Cultures fed daily with a concentrated suspension of Chlorella vulgaris
- Feeding during test: None
Test type:
semi-static
Water media type:
other: Elendt M4 medium
Limit test:
no
Total exposure duration:
48 h
Hardness:
Not specified.
Test temperature:
20 +/- 2 ºC
pH:
7.59 - 8.08
Dissolved oxygen:
8.49 - 9.45 mg/L (91-101% air saturation value, ASV)
Salinity:
Not applicable
Conductivity:
Not specified
Nominal and measured concentrations:
The nominal concentrations tested were 0 (control), 0.019, 0.041, 0.091, 0.20, 0.45 and 1.0 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL (nominal) glass, tall form beakers covered with clear lids
- Test solution volume: 50 mL (nominal)
- Aeration: Not aerated during the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium prepared from trace element stock solutions and reverse-osmosis (RO) water in accordance with OECD Guideline No. 202.

OTHER TEST CONDITIONS
- Intervals of water quality measurement: At the start of the test (0 hours) and at media renewal at 24 hours, 10 mL samples of freshly prepared test media were taken from the control and the test media preparation vessel for chemical analysis.
At 24 and 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media from the test and control groups.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
- Adjustment of pH: None
- Photoperiod: 16-hour light : 8-hour dark
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility. The EL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) at 24 and 48 hours were determined, if possible.

Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.71 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ loading rate WAF
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
(95% confidence limits, 0.62 - 0.81)
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
0.2 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:
EL50
Effect conc.:
> 1 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.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ loading rate WAF
Basis for effect:
mobility
Results with reference substance (positive control):
A positive control study was conducted to determine the 48-hour acute toxicity of the reference substance, potassium dichromate, on the freshwater planktonic crustacean, Daphnia magna. The 48-hour EC50 values for mobility was determined by linear interpolation to be 1.366 mg/L. The 48-hour NOEC value was determined to be 1.0 mg/L. These results were within the expected toxicity range (48-hour EC50: 0.6 - 2.1 mg/L) for this reference substance and all validity criteria were met.
Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8. The 48-hour EL50 value and corresponding 95% confidence limits were calculated using the Trimmed Spearman Kärber method. The 24-hour EL50 value and no observed effect loading rates (NOEL) and the lowest observed effect loading rates (LOEL) were determined empirically.

The results of the range-finding test suggested that the EL50 may be less than 0.10 mg/L loading rate WAF.

Fourth Definitive Test

The results of an initial definitive test showed no immobilisation at the highest loading rate tested of 0.10 mg/L loading rate WAF and the definitive test was therefore repeated.

Final Definitive Test

Chemical Analysis

Samples of test media were analysed at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).Concentrations of the test material were determined by liquid chromatography triple quadrupole mass spectrometry (LC-TQMS) system, using the branched dodecylbenzenamineconstituent as an analytical marker for quantification. The limit of quantification (LOQ) was determined to be 0.0010 mg/L.

Analysis of the freshly prepared test media showed measured concentrations to range from 0.00157 to 0.0937 mg/L at 0 hours and from less than the limit of quantification (LOQ, 0.001 mg/L) of the analytical method to 0.0392 mg/L at 24 hours. The results from the freshly prepared WAFs at 0 and 24 hours showed differences in measured concentrations despite the WAFs being prepared in the same manner. This was attributed to the expected variable water solubility and complex nature of the test substance, as well as the difficulties associated with the preparation of WAFs.

Analysis of the corresponding old media showed measured concentrations to range from <LOQ to 0.0722 mg/L at 24 hours and from <LOQ to 0.0192 mg/L at 48 hours.

Given that the test material is a UVCB substance, and analytical monitoring was limited to the branched dodecylbenzenamine fraction of the test material, the effect concentrations were based on nominal loading rates.

The numbers of immobilised Daphnia magna following exposure to the test substance for 24 and 48 hours are presented below:


Nominal loading rate (mg/L)

Number of
Daphnia magna
exposed

% Immobility
at 24 hours

% Immobility
at 48 hours

Control

20

0

0

0.019

20

0

0

0.041

20

0

5*

0.091

20

0

0

0.20

20

0

0

0.45

20

0

10

1.0

20

0

80

.

 * Single immobilised daphnid not considered not to be biologically significant given that <10% immobilisation observed and no immobilisation observed at the two higher loading rates

Validity criteria fulfilled:
yes
Remarks:
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied.
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated under semi-static conditions and the 48-hour EL50 value was determined to be 0.71 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 0.20 mg/L, based on loading rate WAF.
Executive summary:

The objective of the study was to determine the 48-hour acute toxicity of the test material against the mobility of the freshwater planktonic crustacean, Daphnia magna. The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202, Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

The test material is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with variable water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guidance, the test material was prepared as Water Accommodated Fractions (WAFs).

Based on the results of a range-finding test and an initial definitive test, the definitive test was conducted at nominal loading rates of 0.019, 0.041, 0.091, 0.20, 0.45 and 1.0 mg/L using semi-static test conditions. A concurrent control group was also included. At the start of the test, five juvenile Daphnia magna were added to each test vessel. TheDaphnia magnain each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

Samples of test media were analysed at0 (fresh media), 24 (old and fresh media) and 48 hours (old media). Concentrations of the test material were determined by liquid chromatography triple quadrupole mass spectrometry (LC-TQMS) system, using the branched dodecylbenzenamineconstituent as an analytical marker for quantification. Analysis of the freshly prepared test media showed measured concentrations to range from 0.00157 to 0.0937 mg/L at 0 hours and from less than the limit of quantification (LOQ, 0.001 mg/L) of the analytical method to 0.0392 mg/L at 24 hours. The results from the freshly prepared WAFs at 0 and 24 hours showed differences in measured concentrations despite the WAFs being prepared in the same manner. This was attributed to the expected variable water solubility and complex nature of the test substance, as well as the difficulties associated with the preparation of WAFs. Analysis of the corresponding old media showed measured concentrations to range from <LOQ to 0.0722 mg/L at 24 hours and from <LOQ to 0.0192 mg/L at 48 hours.

Given that the test material is a UVCB and analytical monitoring was limited to the branched dodecylbenzenaminefraction of the test substance, the effect concentrations were based on nominal loading rates.

The 48-hour EL50value was determined to be 0.71 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) was determined to be 0.20 mg/L.

Description of key information

In an OECD 202 study, conducted according to GLP, the acute toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated under semi-static conditions and the 48-hour EL50 value was determined to be 0.71 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 0.20 mg/L, based on nominal loading rate WAF (Smithers Viscient (ESG) Ltd., 2018).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.71 mg/L

Additional information