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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:
9th March - 24th March 2010
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
Analytical monitoring:
Details on sampling:
0, 24 and 48 hours.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Before preparation of each WAF loading rate, volume of five 1L Erlenmeyer’s flasks used in the test were pre-measured up to ground-joint conical stopper used for stopping the flask. This action was performed due to minimize a volatilization process during a preparation of WAFs, because the test substance is volatile liquid.
The other way to minimize a volatilization process was, that the test substance was directly micropipetting into prepared Erlenmeyer’s flasks respecting their pre-measured volume and density of the test substance. Density of the SHALE OILS, LIGHT is 0.7614 g/ml. In the other words micropipetting volumes used for preparation of each loading rate respected density of the test substance and pre-measured volume of each Erlenmeyer flask and corresponded to the required loading rate in mg/l.
After micropipetting of defined volume of the test substance, freshly prepared dilution water was topped up to pre-measured volume of the particular flask and immediately stopped by ground-joint conical stopper.
WAFs were prepared individually at each loading rate used.

Afterwards each of Erlenmeyer flask’s content was stirred with magnetic stirrers (putting into the flask sooner) for the next 24 hours in air-conditioned chamber in the dark at average temperature 20.4 °C for the purpose of the generating of water accommodated fractions for chosen loading rates.
After stirring taking 24 hours, the flasks were transferred to the ultrasonic bath for 30 minutes for the purpose of maximization of the quality of WAFs and attaining of equilibrium and compositional stability over time.
Then a content of each Erlenmeyer flask was passed as quickly as possible through paper filters into new Erlenmeyer flasks for purpose to remove potential visible undissolved part of the test substance. This way prepared filtrates of the dispersions with chosen loading rates of the test substance were distributed into test vessels and tested on daphnids as WAFs. Test vessels were stopped by tight plastic stopper in order to minimize volatility of the WAFs.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: freshwater flea
- Strain: Daphnia magna
- Source: National Reference Centre for Ecotoxicology, within the Public Health Authority of the Slovak Republic
- Age at study initiation (mean and range, SD): < 24 hours
- Weight at study initiation (mean and range, SD): NDA
- Length at study initiation (length definition, mean, range and SD): NDA
- Valve height at study initiation, for shell deposition study (mean and range, SD): NDA
- Peripheral shell growth removed prior to test initiation: NDA
- Method of breeding: Individuals of Daphnia magna are brood in glass breeding stock vessels (volume 1 – 2 litres). As a breeding medium a stood aerated and uncontaminated tap water has been used. This medium is replaced twice per week. Cultivation has been carrying out in the air-conditioned chamber C (Room 208), where constant temperature (20 ± 1 °C), light intensity (ca. 700 lux) and light regime (light/dark 16h/8h) has been maintained.
- Feeding during test: NDA
- Food type: NDA
- Amount: NDA
- Frequency: NDA

24 hours before test was started, a sufficient number of parental daphnids had been replaced from breeding stock vessels containing stood tap water into freshly prepared vessels having already been filled with dilution water (test media). Individuals (newly-born daphnids) being born in these conditions were exposed to the test substance in the day of beginning of the test.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
140 - 250 mg/L
Test temperature:
average temperature recorded was 20.1 ºC
7.62 - 8.43
Dissolved oxygen:
82.9 - 99.4 %
Nominal and measured concentrations:
WAF loading rates of 3.125, 6.25, 12.50, 25.00, and 50.00 mg/L
Details on test conditions:
- Test vessel: glass vials
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 25 mL capacity containing 10 mL test solution
- Aeration: NDA
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): every 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

Dilution water was used as test media. The following chemicals were added:

CaCl2.2H2O at 117.6 mg/L
MgSO4.7H2O at 49.3 mg/L
NaHCO3 at 25.9 mg/L
KCl at 2.3 mg/L

- Adjustment of pH: no
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: ca. 700 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility

- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study NDA
Reference substance (positive control):
Potassium dichromate
48 h
Dose descriptor:
Effect conc.:
9.78 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL of 8.41 - 11.09
Details on results:
TOC analysis:
After 24 hours, the TOC was 81.9 % of the original. After 48 hours, it was 73.7 % of the original concentration. On this basis, it was decided to renew the solutions every 24 hours.
Results with reference substance (positive control):
24 h EiC50 value of the reference compound potassium dichromate was 1.10 mg/l which is within the range specified in ISO 6341 (0.6 – 2.1 mg/l).
Reported statistics and error estimates:
All effective loading rates were calculated by use of Program Probit Analysis, version 2.3, National Swedish Environmental Protection Agency.

Unexpected EiL10 value.

At first sight, it appears that an error has been made with respect to determination of the 24 hour EiL10 value, in that it is lower than the equivalent value at 48 hours, and also lower than 6.25 mg/L, a concentration at which no immobilisation was observed. The explanation lies in the statistical method used to calculate the value.

In this instance, there is high variability in the in the daphnia inhibition recorded after 24 hours exposure, combined with a steep increase between 6.25 - 12.5 mg/L concentration groups (see graph 1). The EiL10 value was determined based on the plot drawn from the immobilisation data. This therefore shows why the EiL10 value is infact a lower concentration than 6.25 mg/L, where no immobilisation had occured.

The fact that the EiL10 value at 24 hours is lower than the equivalent value at 48 hours is again due to the statistical method used, even if common sense would suggest that the reverse should be true. The 48 hour immobilisation data deviated much less from the line of best fit (graphy 2), producing more reliable results than at 24 hours, allowing 95% confidence limits to be determined, something that could not be achieved with the 24 hour data.

The problem with any experiment with living organisms is that the results are always subject to a certain level of variation from what is expected, by the very nature of what you are dealing with. Efforts are made to standardise procedures as much as possible, but incosistencies will always occur with living organisms.

In conclusion, it is the variable immobilisation results obtained experimentally which have led to the unexpected statistical outcome.

Table 1: Immobilisation of Daphnia

Loading Rate (mg/l)

Number of Daphnids tested

Number immobilised after 24 hours

Number immobilised after 48 hours


























Based on recorded inhibition of mobility in time, these effective inhibition loading rates (EiL) were calculated:

24 – hour EiL10 = 5.72 mg/l

95 % confidence interval could not be calculated due to relatively variable inhibition of mobility recorded after 24 – hour exposure)

24 – hour EiL50 = 12.21 mg/l

95 % confidence interval could not be calculated due to relatively variable inhibition of mobility recorded after 24 – hour exposure)

48 - hour EiL10 = 7.06 mg/l

95 % confidence interval: 3.42 – 7.36 mg/l

48 - hour EiL50 = 9.78 mg/l

(95 % confidence interval: 8.34 – 11.33 mg/l)

Lowest loading rate being responsible for 100 % immobilization (EiL100)

After 24 – hours exposure > 50 mg/l (WAF loading rate)

After 48 – hours exposure = 25 mg/l (WAF loading rate)

Highest loading rate being responsible for 0 % immobilization (EiL0)

After 24 – hours exposure = 6.25 mg/l (WAF loading rate)

After 48 – hours exposure = 3.125 mg/l (WAF loading rate)

Validity criteria fulfilled:
see above
The 48 hour EiL50 to daphnia magna of shale oils, heavy was determined to be 9.78 mg/l
Executive summary:

The aim of the study was to determine the loading rates of SHALE OILS, LIGHT inhibiting the mobility of Daphnia magna in a 48-hour semi-static immobilization test. The presented results are based on water accommodated fractions (WAFs) and loading rates of the test substance SHALE OILS, LIGHT, since the test substance belongs to complex multi-component substances only partially soluble in water.

SHALE OILS, LIGHT was tested in loading rates 3.125; 6.25; 12.5; 25 and 50 mg/l. On the basis of the results, it is concluded that the test substance immobilized 50 % of the test animals, the freshwater crustacean Daphnia magna, at a loading rate 12.21 mg/l after 24 - hour exposure (no confidence limits could be determined) and at a loading rate 9.78 mg/l after 48 – hour exposure (confidence limit: 8.41 – 11.09 mg/l) under semi-static conditions.

Also effective inhibition loading rates (EiL) were calculated for 10 % immobilization of Daphnia magna in both time exposures: The 24 – hour EiL10 was calculated on 5.72 mg/l (no confidence limits could be calculated) and the 48 – hour EiL10 was calculated on 7.06 mg/l (confidence limit: 4.66 – 8.43 mg/l).

The lowest loading rate being responsible for 100 % immobilization (EiL100) was determined after 24 – hour exposure at more than 50 mg/l and after 48 – hour exposure at 25 mg/l.

The highest loading rate being responsible for 0 % immobilization (EiL0) was determined after 24 – hour exposure at 6.25 mg/l and after 48 – hour exposure at 3.125 mg/l.

Description of key information

The 48 hour EiL50 to daphnia magna of shale oils, heavy was determined to be 9.78 mg/l (confidence limit: 8.41 – 11.09 mg/l) under semi-static conditions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
9.78 mg/L

Additional information

The study was conducted to recognised test guidelines and following the principles of GLP, but with some reporting deficiencies, and hence was assigned a klimisch score of 2.