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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:
27 June 2012- 13 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
June 2012
Analytical monitoring:
yes
Details on sampling:
Four samples (10 mL) were taken from the freshly-prepared control and test media at 0 and 24 hours that remained after the vessels had been filled. For the expired media at 24 and 48 hours, the contents of the test vessels from each group were pooled and a further four samples (10 mL) were taken for analysis.
On each occasion, two of the samples were analysed and the others were stored in a freezer in case further analysis was required.
Vehicle:
no
Details on test solutions:
The test substance (7.64 mg, corrected for purity) was added to dilution medium (20L, Elendt M4 medium prepared in deionised reverse osmosis water) and stirred for approximately 20 minutes before being used as the test medium at a nominal concentration of 360 µg/L. Test glassware was pre-exposed overnight at the test concentration before use in the test, to minimise loss of BMI by sorption.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name:Daphnia magna
- Strain/clone: Straus
- Source: National Institute for Applied Chemical Research (IRCHA), France
- Age of parental stock: animals less than 24h old were used in the test
- Feeding during test: animals were not fed during the exposure period
- Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 +- 2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.

ACCLIMATION
- Acclimation period: Stock cultures were maintained at the testing lab
- Acclimation conditions: Same as test
- Type and amount of food: Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study
Hardness:
234 and 249 mg/L CaCO3
Test temperature:
19.8 - 20.5 °C for the control test
19.8 - 20.4 °C for the test vessels
pH:
pH 8.06 - 8.25 for control vessels
pH 7.94 - 8.25 for test vessels
Dissolved oxygen:
98-101% ASV for control vessels
99-100% ASV for test vessels
Nominal and measured concentrations:
The range finding test was conducted before the limit of aqueous solubility was identified and employed nominal test concentrations of 0.1, 1, 10 and 100 mg/L. After 48 hours, all of the Daphnia at 100 mg/L were immobile and at 10 mg/L there was 80% immobility. No immobility was observed at 1 or 0.1 mg/L (except for a single daphnid in one replicate at 0.1 mg/L). Based on these results and the stated limit of aqueous solubility, the definitive test employed a single nominal concentration of 360 µg/L (limit test).

The measured concentrations of BMI ranged between 51 and 73% of its nominal value in samples of freshly prepared medium, and between 9 and 16% in samples of expired media (between 18 and 21% of their initial mean measured values). Based on a geometric mean, the overall mean measured level of BMI was 99.4 µg/L, which was used to determine the study end points.
Details on test conditions:
Test area was maintained at 20 +/- 2ºC during the definitive test. The temperature of the test area was continuously monitored in an additional vessel containing the same volume of dilution medium. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. No supplementary aeration was employed and no feed was given during the exposure period.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
99.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 99.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 99.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No immobilisation or sub-lethal adverse effects on the Daphnia were noted.

Table1           Measured concentrations

Nominal

concentration

(µg/L)

Measured BMI concentrations (µg/L)

Overall

mean*

(µg/L)

0 hours [Fresh]

%N

24 hours [Expired]

%N

% t0

24 hours [Fresh]

%N

48 hours [Expired]

%N

% t24

 

 

 

 

 

 

 

 

 

 

 

 

Control

nd

-

nd

-

-

nd

-

nd

-

-

-

360

264

73

47.8

13

21

183

51

41.0

11

13

99.4

246

68

59.0

16

220

61

31.4

9

 

 

 

 

 

 

 

 

 

 

 

 

 

nd          None detected (less than the limit of detection of 2 µg/L).

%N        Measured concentration expressed as a percentage of the nominal concentration.

%t          Mean measured concentration (expired medium at 24 or 48 hours) expressed as a percentage of the mean starting concentration (fresh medium at 0 or 24 hours).

*             Geometric mean.

 

 

 

 

 

Table2           Environmental parameters

Nominal

BMI concentration (µg/L)

Measured BMI concentration (µg/L)

Temperature (°C)

pH

Dissolved oxygen
 (%ASV)

0 hours

24 hours

24 hours

48 hours

0 hours

24 hours

24 hours

48 hours

0 hours

24 hours

24 hours

48 hours

Fresh

Expired

Fresh

Expired

Fresh

Expired

Fresh

Expired

Fresh

Expired

Fresh

Expired

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Control

nd

19.8

20.5

19.8

19.7

8.06

8.25

8.19

8.24

98

101

100

100

360

99.4

19.8

20.4

19.9

19.5

7.94

8.24

8.17

8.25

99

100

100

99

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

nd          None detected (less than the limit of detection of 2 µg/L).

ASV       Air saturation value(100% ASV = 9.09 mgO2/L at 20ºC).

The total hardness and alkalinity of two batches of Elendt M4 medium used were 234 & 249 and 49 & 39 mg/L as CaCO3, respectively.

Continuous monitoring of an additional vessel containing dilution medium = 18.5 to 19.3°C.

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria
Conclusions:
BMI was not found to be acutely toxic to Daphnia magna at measured concentrations up to 99.4 ug/L, which was considered to reflect the maximum achievable concentrations within the limit of solubility of the test substance.
Consequently, the 48-hour EC50 could not be calculated but must be >99.4 ug/L and the “no-observed effect concentration” was 99.4 ug/L.
Executive summary:

A Daphnia magna immobilisation test was undertaken under semi-static exposure conditions over a period of 48 hours, with renewal of the media at 24 hours. The study was conducted in accordance with EC Method C2 “Daphnia sp. Acute Immobilisation Test” and OECD Guideline 202, “Daphnia Acute Immobilisation Test” (2004) and GLP conditions.

A group of twenty Daphnia, less than 24 hours old, was exposed for 48 hours to BMI at a single nominal concentration of 360 µg/L (stated limit of aqueous solubility). The test medium was prepared in Elendt M4 medium by the direct addition of the test substance to the dilution medium; stirring was used to aid dissolution. To minimise losses by sorption, the test glassware was pre-exposed to BMI at the test concentration overnight before use.

The measured concentrations of BMI ranged between 51 and 73% of its nominal value in samples of freshly prepared medium, and between 9 and 16% in samples of expired media (between 18 and 21% of their initial mean measured values). Based on a geometric mean, the overall mean measured level of BMI was 99.4 µg/L, which was used to determine the study end points.

Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. No immobilisation or any sub-lethal effects on the Daphnia were noted, therefore the EC50value was considered to be > 99.4 µg/L. The no observed effect concentration (NOEC) was 99.4 µg/L, and the lowest observed effect concentration was (LOEC) >99.4 µg/L. The study met all validity criteria and is considered to be reliable without restrictions.

Description of key information

48 hr NOEC for Daphnia Magna 99.4 µg/L, 48 hr EC50 and LOEC considered to be >99.4 µg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
99.4 µg/L

Additional information

A Daphnia magna immobilisation test was undertaken under semi-static exposure conditions over a period of 48 hours, with renewal of the media at 24 hours. The study was conducted in accordance with EC Method C2 “Daphnia sp. Acute Immobilisation Test” and OECD Guideline 202, “Daphnia Acute Immobilisation Test” (2004) and GLP conditions.

A group of twenty Daphnia, less than 24 hours old, was exposed for 48 hours to BMI at a single nominal concentration of 360 µg/L (stated limit of aqueous solubility). The test medium was prepared in Elendt M4 medium by the direct addition of the test substance to the dilution medium; stirring was used to aid dissolution. To minimise losses by sorption, the test glassware was pre-exposed to BMI at the test concentration overnight before use.

The measured concentrations of BMI ranged between 51 and 73% of its nominal value in samples of freshly prepared medium, and between 9 and 16% in samples of expired media (between 18 and 21% of their initial mean measured values). Based on a geometric mean, the overall mean measured level of BMI was 99.4 µg/L, which was used to determine the study end points.

Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. No immobilisation or any sub-lethal effects on the Daphnia were noted, therefore the EC50value was considered to be > 99.4 µg/L. The no observed effect concentration (NOEC) was 99.4 µg/L, and the lowest observed effect concentration was (LOEC) >99.4 µg/L.The study met all validity criteria and is considered to be reliable without restrictions.