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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:
26 February 2018 to ****
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:
-Purity: Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
-Description: Dark Green Liquid

Analytical monitoring:
yes
Remarks:
Given that the test substance is a multi-constituent substance and analytical monitoring was limited to the dithiophosphate fraction (without molybdenum) of the test substance, the results were based on nominal loading rates.
Details on sampling:
- Concentrations: Control, 1.0, 3.2, 10, 32, and 100 mg/L loading rate WAF
- Sampling method: Aqueous samples (10 mL) of freshly prepared test media (0 hour) and pooled expired test media (48 hours) were collected in duplicate, mixed with acetonitrile, then diluted further with Elendt M4 media:acetonitrile (1:1, v/v) as required to bring the response within the calibration range.
- Sample storage conditions before analysis: Not applicable
Vehicle:
no
Details on test solutions:
The substance is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with low 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 guideline, the test solutions were prepared as Water Accommodated Fractions (WAFs).

At the start of the test, the test substance was weighed onto a glass slides and placed in 1000 mL of Elendt M4 test medium. The preparations were stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparations were allowed to settle for ca 1 hour, after which the aqueous phase was removed by careful mid-depth siphoning to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF. A control group was prepared in the same manner without the addition of test substance.
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
- 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:
static
Water media type:
other: Elendt M4 medium
Total exposure duration:
48 h
Hardness:
Not specified.
Test temperature:
20 ± 2ºC; constant within 1ºC in the test vessels. The temperature monitored continuously in an adjacent vessel varied by more than 1ºC, but was not considered to have adversely affected the integrity of the test since no immobilization was observed.
pH:
7.72 to 8.28
Dissolved oxygen:
8.55 – 9.22 mg/L (93 – 97% air saturation value)
Salinity:
Not specified.
Conductivity:
Not specified.
Nominal and measured concentrations:
See Table 2 (below).
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 specified
- 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: 0 and 48 hours
- Adjustment of pH: none
- Photoperiod: 16-hour light : 8-hour dark
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The EL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) were estimated empirically by observation of the data.

VEHICLE CONTROL: not applicable

RANGE-FINDING STUDY
- Test concentrations: 0, 1.0, 10 and 100 mg/L (duplicate vessels for each)
- Results used to determine the conditions for the definitive study: See immobilization data in Table 1 (below).
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rate WAF
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
other: nominal mg/L loading rate WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
other: nominal mg/L loading rate WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
other: nominal mg/L loading rate WAF
Basis for effect:
mobility
Details on results:
- Behavioral abnormalities: none
- Immobilization in negative control: none
- Other adverse effects in negative control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All WAF exposure preparations were colorless solutions indicating that no undissolved test substance was present. The apparent decrease in concentration at the 100 mg/L loading rate WAF after 48 hours, however, could be attributed to the presence of microscopic particles or micelles, which were not readily visible and which may have settled out of solution over the test period, given the stability observed in the remaining WAFs. This was considered not to have affected the integrity of the test given that no immobilization was observed at any loading rate during the test. Given that the test substance is a multi-constituent substance, the results were based on nominal loading rates.
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
- Reference substance: potassium dichromate
- Test design: same test organism, medium, exposure conditions and replication as this definitive test
- Results with reference substance valid? Yes; EC50 result was within the expected range state in the OECD Guideline No. 202 (0.6 - 2.1 mg/L).
- Relevant effect levels: 48-hour EC50
- Dose-response test: yes; treatment concentrations were 0 (control), 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- EC50: 1.366 mg/L
- NOEC: 1.0 mg/L
Reported statistics and error estimates:
Statistical analysis was not performed given that no immobilisation was observed during the test.

Table 1: Immobilization in rangefinding test

Nominal

Cumulative immobilised Daphnia magna

(initial population: 5 Daphnia per replicate)

loading rate

24 hours

48 hours

%
Immobility

(mg/L)

R1

R2

R1

R2

Control

0

0

0

0

0

1.0

0

0

0

1

10

10

0

0

1

0

10

100

0

0

0

0

0

R = Replicate

 

 Table 2: Analysis of test media during the definitive test

Nominal

Measured Concentration* (mg/L)

loading rate

0 Hours

48 Hours

(mg/L)

(New)

(Old)

Control

<LOQ

<LOQ

1.0

0.0262

0.0299

3.2

0.0277

0.0161

10

0.135

0.126

32

0.229

0.200

100

0.769

0.237

 * Based on quantitation of dithiophosphate fraction (without molybdenum)

   LOQ = Limit of quantification (0.01 mg/L)

Table 3: Immobilization in definitive test

Nominal loading rate (mg/L)

Number of
Daphnia magna
exposed

% Immobility
at 24 hours

% Immobility
at 48 hours

Control

20

0

0

1.0

20

0

0

3.2

20

0

0

10

20

0

0

32

20

0

0

100

20

0

0

Validity criteria fulfilled:
yes
Remarks:
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.
Conclusions:
No immobilization was observed after 48 hours of exposure. The 48-hour EL50 value was therefore considered to be >100 mg/L loading rate WAF. The test showed no toxicity at the limit of solubility of the test substance in the test media.
Executive summary:

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202. Test solutions were prepared as Water Accommodated Fractions (WAFs) based on the recommendations of OECD Series on Testing and Assessment, No. 23. Based on the results of a range-finding test the definitive test was conducted at nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L under static conditions, with a concurrent control group.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed for mobility at 24 and 48 hours. The Daphnia magna were not fed during the test.

Samples of test media were analyzed at0 (fresh media) and 48 hours (old media) usinghigh performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) based on quantitation of the dithiophosphatefraction (without molybdenum) of the test substance. Analysis of the test media confirmed measured concentrations to range from 0.0262 to 0.769 mg/L at 0 hours and from 0.0299 to 0.237 mg/L at 48 hours. Given that the test substance is a multi-constituent substance and analytical monitoring was limited to the dithiophosphatefraction (without molybdenum) of the test substance, the effect concentrations were based on nominal loading rates.

No immobilization was observed over the 48-hour exposure period. The 48-hour EL50 value was therefore determined to be >100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 100 mg/L loading rate WAF. The test showed no toxicity at the limit of solubility of the test substance in the test media.

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Description of key information

In an OECD 202 study, conducted according to GLP, the acute toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated under static conditions and the 48 -Hour EC50 value was determined to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 100 mg/L loading rate WAF (Smithers Viscient (ESG) Ltd, ****).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

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