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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:
21 January to 16 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to recognised guidelines
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
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:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal concentrations: 4.27, 9.39, 20.7, 45.5 and 100 mg/L + controls.

- Sampling method: At the start of the definitive test, two samples (10 mL) were taken from the freshly-prepared control and test media (pH-adjusted and non-adjusted media). After 48 hours, the contents of the test vessels from each group were pooled and further samples were
taken for analysis.

- Sample storage conditions before analysis: On each occasion, one of the samples was analysed and the other was stored in a freezer in case further analysis was required.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: The test substance (500 mg) was dissolved in dilution medium (5 L) in a volumetric flask. The contents of the flask were mixed and the pH was measured. If the pH was 9 or higher, an aliquot (1 L) of the medium was removed; the pH of this portion was adjusted to 7.6 ± 0.2 (using 0.1 M HCl) and used as the pH-adjusted media. The remaining non-adjusted media was then either used directly at the highest test concentration or diluted to provide the test media at the four lower concentrations. At each concentration where the pH of the medium was 9 or higher, an aliquot of the non-adjusted medium was adjusted as detailed above. pH adjustment was required at 20.7, 45.5 and 100 mg/L. Nominal test levels: contol, 4.27, 9.39, 20.7, 45.5, 100 mg/L

- Eluate: not applicable

- Differential loading: not applicable

- Controls: Dilution medium (Elendt M4)

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable

- Concentration of vehicle in test medium (stock solution and final test solution): not applicable ('vehicle' was dilution medium)

- Evidence of undissolved material (e.g. precipitate, surface film, etc): none reported
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Straus
- Source: the National Institute for Applied Chemical Research (IRCHA), France.
- Age at study initiation (mean and range, SD): <24 h
- Method of breeding: parthenogenesis
- Feeding during test: none

ACCLIMATION
- Acclimation period: none
- Acclimation conditions (same as test or not): as test
- Type and amount of food: unicellular green algae, Pseudokirchneriella subcapitata/0.1 to 0.2 mg carbon per daphnid except during the initial three days when a slightly lower ration was given.
- Feeding frequency: once per day
- Health during acclimation (any mortality observed): not stated


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
266 mg/L as calcium carbonate
Test temperature:
19.1 - 20.2 ºC (Allowed range - 20 +/- 2ºC)
pH:
At nominal concentrations of 20.7, 45.5 and 100 mg/L, the pH of the media following the addition of the test substance exceeded that
recommended for this type of test (6 – 9). Therefore at these levels an additional set of media was prepared in which the media was adjusted to levels typically used in this type of test. The high pH at these concentrations also caused the pH to vary by more than 1.5 units during the test although this does not invalidate the test as all validity criteria were met. At 48 hours, the pH of the non-adjusted media had decreased to within the range stated in the protocol.

7.65 - 10.01
7.68 - 7.96 (pH adjusted vessels)
Dissolved oxygen:
As % air saturation value: 101 - 106
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal: 4.27 9.39 20.7 45.5 100 non pH adjusted media
Measured: 4.41 9.06 21.1 48.6 97.8
Nominal: 20.7 45.5 100 pH adjusted media
Measured: 21.0 47.0 100
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): glass dishes loosely covered
- Material, size, headspace, fill volume: glass 100 mL. Headspace and fill volume not stated
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): not applicable
- 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 as vehicle was test medium
- Biomass loading rate: not stated

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of test/dilution water: The test organisms were maintained and the tests conducted in Elendt M7 medium prepared using deionised, reverse osmosis water
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: pH of test solutions was maintained below 9
- Photoperiod: 16 hours light : 8 dark
- Light intensity: not stated other than subdued at beginning and end of each light phase

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation of daphnids


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: The range finding test employed nominal test concentrations of 0.1, 1, 10 and 100 mg/L. At 10 and 100 mg/L, the pH was measured to be 9 or higher. A pH of 9 or higher is outside of the range stated in the protocol (6 – 9) and could significantly affect the survival of Daphnia magna therefore a second batch of media were prepared in which the pH was adjusted. In the non-adjusted media after 48 hours, all of the Daphnia at 100 mg/L were immobile and all Daphnia were mobile at 10 mg/L. In the pH-adjusted media after 48 hours, 80% of the Daphnia at 100 mg/L were immobile and all Daphnia were mobile at 10 mg/L. Based on these results, the definitive test employed nominal concentrations of:
Nominal concentrations: 4.27, 9.39, 20.7, 45.5 and 100 mg/L
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material - pH not adjusted
Basis for effect:
mobility
Remarks on result:
other: 95% CL 41.1 53.1
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
37.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material - pH not adjusted
Basis for effect:
mobility
Remarks on result:
other: 95% CL 34.0, 48.6
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
21.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material - pH not adjusted
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
68.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material - pH adjusted
Basis for effect:
mobility
Remarks on result:
other: 95% CL 53.3 95.4
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
62.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material - pH adjusted
Basis for effect:
mobility
Remarks on result:
other: 95% CL 49.1 84.1
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
21 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material - pH adjusted
Basis for effect:
mobility
Details on results:
- Other biological observations: none
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
48 h EC50 (pH non-adjusted): 37.5 mg/L (95% CL 34.0 - 48.6 mg/L). NOEC: 21.1 mg/L
48 h EC50 (pH adjusted): 62.8 mg/L (95% CL 49.1 - 84.1 mg/L). NOEC: 21.0 mg/L

EC50values and their 95% confidence limits were calculated using the LD50 application of the SAFEStat system, SAS 8.2 (SAS Institute, 1999).

Measured Concentrations

Non-adjusted media

 

Nominal conc.

Measured MODA concentrations (mg/L)

Overall

(mg/L)

0 hours

%N

48 hours

%N

% ti

mean*

Control

nd

-

nd

-

-

-

4.27

4.70

110

4.12

96

88

4.41

9.39

9.59

102

8.53

91

89

9.06

20.7

22.6

109

19.6

95

87

21.1

45.5

49.8

109

47.4

104

95

48.6

100

97.7

98

97.8

98

100

97.8

 

 

pH-adjusted media

 

Nominal conc.

Measured MODA concentrations (mg/L)

Overall

(mg/L)

0 hours

%N

48 hours

%N

% ti

mean*

20.7

22.2

107

19.7

95

89

21.0

45.5

51.2

113

42.8

94

84

47.0

100

99.8

100

101

101

101

100

 

nd : none detected (< 0.5 mg/L).

% ti : mean measured concentration after 48 hours expressed as a percentage of the mean starting

concentration.

%N : mean measured concentration expressed as a percentage of the nominal concentration.

* : arithmetic mean.

At all concentrations, the intended exposure levels of MODA were adequately achieved (between 91 and 113% of their nominal values) and maintained during the test (between 84 and 101% of their starting values) irrespective of whether the pH of the media was adjusted. The overall mean measured levels of MODA were 4.41, 9.06, 21.1, 48.6 and 97.8 mg/L in non-adjusted media and 21.0, 47.0 and 100 mg/L in media where the pH had been adjusted.

Immobility:

After 48 hours in the non-adjusted media, the lowest measured concentration resulting in 100% immobility was 97.8 mg/L and the highest concentration at which no immobilisation occurred was 21.1 mg/L. After 48 hours in the pH-adjusted media 70% of the Daphnia were immobile at 100 mg/L and the highest concentration at which no immobilisation occurred was 21.0 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 of MODA for the immobilisation of Daphnia magna in non-adjusted media was 37.5 mg/L (95% confidence limits of 34.0 and 48.6 mg/L) and in pH-adjusted media it was 62.8 mg/L (95% confidence limits of 49.1 and 84.1 mg/L).

The ‘no-observed effect concentration’ of MODA with Daphnia magna was 21.1 and 21.0 mg/L respectively, in non-adjusted and pH-adjusted media.

These results suggest that the pH of the test media, increased by the presence of the test substance, was partially responsible for the toxicity
observed.

Description of key information

The 48-hour EC50 of MODA for the immobilisation of Daphnia magna in non-adjusted media was 37.5 mg/L (95% confidence limits of 34.0 and 48.6 mg/L) and in pH-adjusted media it was 62.8 mg/L (95% confidence limits of 49.1 and 84.1 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
62.8 mg/L

Additional information

The ‘no-observed effect concentration’ of MODA with Daphnia magna was 21.1 and 21.0 mg/L respectively, in non-adjusted and pH-adjusted media. These results suggest that the pH of the test media, increased by the presence of the test substance, was partially responsible for the toxicity observed.