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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 12 Jan 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Department of Health of the government of the United Kingdom
Specific details on test material used for the study:
Storage conditions: ambient temperature (10 to 30 °C) "
Analytical monitoring:
Details on sampling:
- Concentrations and sampling method: 10 mL samples were collected at timepoints 0, 24 and 48 h from both the freshly prepared and the old media for chemical analysis. Old test media were pooled prior to sampling. Additionally a sample from the 100 mg/L test concentration prepared without organisms was taken for analysis.

Details on test solutions:
- Method: 500 mg of test substance was added to 1000 mL of medium. The solution was filtered through a 0.45 μm filter. A sample of the filtrate was taken for chemical analysis. Based on the measured concentration of the filtrate, a dilution was prepared in medium resulting in the 100 mg/L test concentration. From this concentration a serial dilutions was performed in order to prepare the remaining test concentrations.
- Evidence of undissolved material and development of method for preparation of test solutions: prior to the test beginning it was observed that the test substance formed a dispersion in the test medium. Therefore the test medium with test substance was filtered. However, the test substance analysis showed that no test substance was present in the filtered sample. Therefore a solubility test and a medium preparation trial was performed:
- SOLUBILITY TEST: nominal concentrations of 100, 500 and 1000 mg/L were prepared and filtered through a 0.45 μm filter. Duplicates were chemicaly analysed.
- MEDIA PREPARATION TRIAL: A 500 mg/L stock solution was prepared, in duplicate, and filtered through a 0.45 μm filter. Duplicate samples were chemicaly analysed. Analysis was repeated after 24 and 48 h. Based on these results, the test solutions for the actual test were prepared as described under `method´.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Age of parental stock: < 24 h old
- Feeding during test: no

- Acclimation conditions: 24 h before a test was set up, juveniles present in the cultures were removed. Over the next 24 h, juveniles for use in the test were removed from the culture and transferred to fresh culture medium. Jjuveniles were then left for at least 1 h. Actively swimming individuals were chosen for the test.

- Type and amount of food: concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures
- Feeding frequency: daily

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Test temperature:
18.6 - 19.8
7.35 - 7.69
Dissolved oxygen:
87 - 98% of air saturation value
8.39 - 9.58 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 10, 18, 32, 56 and 100 mg/L
Measured: < LOQ, 6.5, 9.3, 11, 15 and 33 mg/L (time-weighted mean measured)
Details on test conditions:
- Test vessel: glass tall form beakers covered with a lid
- Fill volume: 50 mL
- Renewal rate of test solution: 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per abiotic control (replicates): 1 vessel for the 100 mg/L test item concentration

- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH, dissolved oxygen concentration and temperature were determined in freshly prepared test media and in the old media at 24 and 48 h.

- Adjustment of pH: no
- Photoperiod: 16-hour light: 8-hor dark (fluorescent lighting)

EFFECT PARAMETERS MEASURED: mobility was observed after 24 and 48 h

- Test concentrations: 1, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: no short term toxicity was observed with an EC50 (48 h) of > 100 mg/L (nominal)
Reference substance (positive control):
48 h
Dose descriptor:
Effect conc.:
> 33 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Mortality of control: no mortalities
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: although the initial 500 mg/L stock solution was a colourless solution, the 10 mg/L test concentration was observed to be very slightly cloudy, the 18-56 mg/L test concentrations were slightly cloudy and the 100 mg/L test concentration was cloudy, non-homogenous dispersions with some precipitates. This was contrary to the observations from the range-finding/limit test and the media preparation test where prepared concentrations were observed to be colourless solutions. It was considered possible that the test substance was reacting with the salts dissolved in the test media. This was considered not to affect the validity of the test given that no immobilisation was observed during the test which was consistent with the range finding test. The control was a colourless solution throughout the test.


Table 1: Results from the solubility test.

Initial concentration
Sample number Measured concentration
Mean measured
concentration (mg/L)
100 1 <LOQ <LOQ
2 <LOQ
500 1 280 258
2 236
1000 1 329 338
2 347

LOQ – Limit of quantification assessed as 5 mg/L

Based on the results of the solubility test, it was considered appropriate to prepare the test solutions from an initial 500 mg/L stock solution followed by filtration through a 0.45 μm filter. The resulted filtrate was analysed to determine the concentration prior to dilution and give the highest test concentration of 100 mg/L.


Table 2: Test item concentrations in the media during the test.

0 h
(New media)
% nominal 24 h
(Old media)
% nominal 24 h
(New media)
% nominal 48 h
(Old media)
% nominal Time weighted
Control <LOQ  - <LOQ  - <LOQ  - <LOQ  -  -
10 10.25 103 5.95 60 8.93 89 <LOQ  - 6.5
18 17.94 100 5.13 29 20.13 112 <LOQ  - 9.3
32 26.55 83 <LOQ  - 36.31 113 <LOQ  - 11
56 43.3 77 <LOQ  - 51.52 92 <LOQ  - 15
100 82.28 82 8.46 8 104.89 105 5.35 5 33
100* NA NA 10.18 10 NA NA 8.62 7 NA

* Vessel without test organisms

** Where measured concentrations were <LOQ, a value of half LOQ (2.5 mg/L) was used to calculate the time-weighted mean measured concentration

The 100 mg/L test concentration without test organisms, gave similar results to the samples from media containing test organisms. This indicated that the decline of the test substance in the medium during the test period was possibly due to chemical and not biological reasons.

Due to the decrease of the measured concentrations over a 24-hour period, it was considered appropriate to base the results on time-weighted mean measured test concentrations.


Table 3: Numbers of immobilised Daphnia magna after 48 h exposure.

Time-weighted mean
measured concentration
Immobility at 48 h
Control Control 0
10 6.5 0
18 9.3 0
32 11 0
56 15 0
100 33 0

Table 4: Further toxicity results.

Time-weighted mean measured concentration (mg/L)
LOEC (48 h) >33
NOEC (48 h) ≥33
Validity criteria fulfilled:
control immobility was ≤10% and dissolved oxygen was ≥3 mg/L
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
48 h
Dose descriptor:
Effect conc.:
> 33 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:

Description of key information

EC50 (48 h) > 33 mg/L (meas. TWA, OECD 202) based on read across from CAS 6009-70-7

Key value for chemical safety assessment

Additional information

No data are available on the toxicity of dipotassium oxalate anhydrate (CAS 583-52-8) to aquatic invertebrates. Therefore, a read across was performed to the analogues substance diammonium oxalate monohydrate (CAS 6009-70-7). Details on the read across justification are provided in section 13 of the IUCLID dossier.

The study was conducted according to the OECD guidance 202 and GLP standards (reference: 6.1.3-1). In a semi-static system the test organism Daphnia magna was exposed for 48 h to nominal test concentrations of 0 (control), 10, 18, 32, 56 and 100 mg/L (corresponding to: < LOQ (control), 6.5, 9.3, 11, 15 and 33 mg/L TWA). An analytical monitoring was performed via LC-TOF/MS. Analysis of the test media samples was conducted at 0 (fresh media), 24 (old and fresh media) and 48 h (old media).The recovery of the test substance was between < LOQ and 60% after 24 h incubation. Therefore the results are expressed in means of the time weighted average. No toxic effects were observed up to the highest tested concentration resulting in an EC50 (48 h) of > 33 mg/L (meas. TWA).

Based on the results of the read across substance, no toxic effects are expected by dipotassium oxalate to aquatic invertebrates.