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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:
15 May 2016 - 14 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Version / remarks:
December 14, 2000
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from all test concentrations and the control at the start and the end of both 24-hour renewal periods.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test item was not completely soluble in test medium at the loading rates initially prepared. All test solutions were prepared separately and started with loading rates ranging from 0.46 to 100 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for approximately 1 - 1½ hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820).
Source In-house laboratory culture with a known history.
Age at study initiation: daphnids not older than 24 hours.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Semi-static with renewal of test solutions after 24 hours
Post exposure observation period:
none
Hardness:
not reported
Test temperature:
20-21°C
pH:
7.7 - 8.0
Dissolved oxygen:
8.2 - 9.1 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Geometric means of the test concentrations were determined as (A+B)/2, where A is the geometric mean concentration of Geraldehyde measured in the samples taken at the start (t = 0) and after 24 hours in old (t = 24h, old) medium and B is he geometric mean concentration of Geraldehyde measured in the samples taken in fresh medium (t = 24h, fresh) and at the end of the test (t = 48h).

Analysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.16, 0.37, 0.85, 2.4 and 4.7 mg/L in the WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. The lowest concentration decreased to 9.6% of initial at the end of the 24-hour renewal period, while all higher concentrations remained stable (80-120% of initial). Analysis of the samples taken at the start of the second renewal period showed slightly higher measured concentrations, i.e. 0.18, 0.53, 1.3, 2.9 and 6.2 mg/L in the WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. The lowest concentration decreased only slightly to 76% of initial at the end of the 24-hour renewal period, while all higher concentrations remained fairly stable (79-89% of initial). Given this result, the EC50 values were expressed in terms of the average exposure concentrations calculated as described above: 0.11, 0.42, 1.0, 2.4 and 5.4 mg/L at nominal concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg/, respectively.
Details on test conditions:
TEST SYSTEM
The tests were performed in 60 mL, all-glass test vessels. Since the test item was treated as volatile the study was performed in air-tight vessels.

* Screening test:
- Three test concentrations (1.0, 10 and 100 mg/L) plus one control each in quadruplicate (control and 100 mg/L) or duplicate (1.0 and 10 mg/L) with 5 animals/test vessel (i.e. 10 animals/concentration).

* Main test
- Five test concentrations (WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L) plus one control each in 4-fold with 5 animals/test vessel (i.e. 20 animals/concentration).
- Loading rate: ca. 12 mL test solution per organism
- Test vessels were maintained at 20 °C under a photoperiod of 16 hours light : 8 hours dark and without supplementary aeration or feeding during the 48 hour exposure period.
- pH and dissolved oxygen were measured at the start and the end of both renewal periods for all concentrations and the control, the temperature of medium was recorded continuously in a temperature control vessel.


EFFECT PARAMETERS MEASURED:
Daphnia were considered to be immobile if they were unable to swim within 15 seconds after gentle agitation of the test vessel.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (tested between 18 and 20 July 2016)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Endpoints were based on analytically confirmed geometric mean measured concentrations.
Details on results:
In tIn the range finding test, at nominal concentrations of 0 (control), 1, 10 and 100 mg/L, respectively, immobility was 0%, 40%, 100% and 100% after 48 hours.

In the final test, at average exposure concentrations of 0 (control), 0.11, 0.42, 1.0, 2.4 and 5.4 mg/L, respectively, immobility was 0%, 0%, 0%, 5%, 100% and 100% after 24 hours, and 0%, 0%, 0%, 95%, 100% and 100% after 48 hours.

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Vailidity criteria were fulfilled, i.e. <10% immobility or trapping at the water surface in the control and dissolved oxygen concentration at the end of the test was >= 3 mg/L.
Results with reference substance (positive control):
The 48h-EC50 was 0.39 mg/L with a 95% confidence interval ranging from 0.33 to 0.44 mg/L. The historical ranges for the 48h-EC50 lie between 0.28 and 0.90 mg/L. The observed 48h-EC50 corresponds with this range.
Reported statistics and error estimates:
The 24-h EC50 was 1.5 mg/L (95% confidence interval could not be determined)
The 48-h EC50 was 0.73 mg/L (95% confidence interval 0.63-0.82 mg/L).
The p(Chi2) value for the overall fit of the 48-h data was 0.85900.
The p(F) value for the slope of the 48-h data was 0.00100.

Table 1 shows the responses recorded during the final test. After 48 hours of exposure ≥ 95% of the daphnids exposed to average concentrations of 1.0 mg/L and higher were immobilized. No immobility was observed in the lower concentrations and the control. The responses recorded in this test allowed for reliable determination of an EC50.

Table1 Incidence of immobility in the final test

Time (h)

Replicate

Average concentration Geraldehyde (mg/L)

Control

0.11

0.42

1.0

2.4

5.4

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0 [2]

5 [1]

5

B

0

0

0

1

5

5

C

0

0

0

0

5

5

D

0

0

0

0

5

5

Total immobilised

0

0

0

1

20

20

Effect %

0

0

0

5

100

100

 

 

 

 

 

 

48

A

0

0

0

4

5

5

B

0

0

0

5

5

5

C

0

0

0

5

5

5

D

0

0

0

5

5

5

Total immobilised

0

0

0

19

20

20

Effect %

0

0

0

95

100

100

[ ]: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Table 2 WAF’s, measured and average exposure concentrations

Geraldehyde, WAF at x mg/L

Measured concentration (mg/L)

Average exposure concentration (mg/L)

t=0h (fresh)

t=24h

(old)

t=24h (fresh)

t=48h (old)

0.46

0.159

0.015

0.184

0.141

0.11

1.0

0.374

0.299

0.531

0.475

0.42

2.2

0.851

1.02

1.25

1.01

1.0

4.6

2.40

2.08

2.92

2.31

2.4

10

4.70

5.36

6.15

5.26

5.4

Table 3 Concentrations of the test item in test medium - final test

Time of sampling
[hours]

Loading rate1

[mg/L]

Concentration
analysed
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

(fresh)

0.46

0.159

 

 

1.0

0.374

 

 

2.2

0.851

 

 

4.6

2.40

 

 

10

4.70

 

 

 

 

 

24

0

n.d.

n.a.

(old)

0.46

0.0152

9.6

 

1.0

0.299

80

 

2.2

1.02

120

 

4.6

2.08

87

 

10

5.36

114

 

 

 

 

24

0

n.d.

 

(fresh)

0.46

0.184

 

 

1.0

0.531

 

 

2.2

1.25

 

 

4.6

2.92

 

 

10

6.15

 

 

 

 

 

48

0

n.d.

n.a.

(old)

0.46

0.141

76

 

1.0

0.475

89

 

2.2

1.01

81

 

4.6

2.31

79

 

10

5.26

86

 

 

 

 

1  A water accommodated fraction (WAF) prepared at the loading rate.

2  Estimated value, calculated by extrapolation of the calibration curve.

n.d.    Not detected.

n.a.     Not applicable.

Validity criteria fulfilled:
yes
Remarks:
<10% immobility or trapping at the water surface in the control; at t=48 h dissolved O2 concentration >=3 mg/L
Conclusions:
The 48 h EC50 (immobilisation) value with Daphnia magna is 0.73 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted under semi-static conditions in accordance with OECD Guideline for Testing of Chemicals No. 202 and in compliance with GLP.

All test solutions were prepared separately and started with loading rates ranging from 0.46 to 10 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for approximately 1½ hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L. The total exposure period was 48 hours and test solutions were renewed daily. Samples for analytical confirmation of exposure concentrations were taken at the start and the end of both renewal periods. Analysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.16, 0.37, 0.85, 2.4 and 4.7 mg/L in the WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. The lowest concentration decreased to 9.6% of initial at the end of the 24-hour renewal period, while all higher concentrations remained stable (80-120% of initial). Analysis of the samples taken at the start of the second renewal period showed slightly higher measured concentrations, i.e. 0.18, 0.53, 1.3, 2.9 and 6.2 mg/L in the WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. The lowest concentration decreased only slightly to 76% of initial at the end of the 24-hour renewal period, while all higher concentrations remained fairly stable (79-89% of initial). Given this result, the EC50 values were expressed in terms of the average exposure concentrations that were calculated to be: 0.11, 0.42, 1.0, 2.4 and 5.4 mg/L.

At average exposure concentrations of 0 (control), 0.11, 0.42, 1.0, 2.4 and 5.4 mg/L, respectively, immobility was 0%, 0%, 0%, 5%, 100% and 100% after 24 hours, and 0%, 0%, 0%, 95%, 100% and 100% after 48 hours. The 48h-EC50 was 0.73 mg/L (95% confidence interval between 0.63 and 0.82 mg/L).

Description of key information

The EC50 for Daphnia magna (OECD TG 202): 0.73 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.73 mg/L

Additional information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted under semi-static conditions in accordance with OECD Guideline for Testing of Chemicals No. 202 and in compliance with GLP.

All test solutions were prepared separately and started with loading rates ranging from 0.46 to 10 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for approximately 1½ hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L. The total exposure period was 48 hours and test solutions were renewed daily. Samples for analytical confirmation of exposure concentrations were taken at the start and the end of both renewal periods. Analysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.16, 0.37, 0.85, 2.4 and 4.7 mg/L in the WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. The lowest concentration decreased to 9.6% of initial at the end of the 24-hour renewal period, while all higher concentrations remained stable (80-120% of initial). Analysis of the samples taken at the start of the second renewal period showed slightly higher measured concentrations, i.e. 0.18, 0.53, 1.3, 2.9 and 6.2 mg/L in the WAFs at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. The lowest concentration decreased only slightly to 76% of initial at the end of the 24-hour renewal period, while all higher concentrations remained fairly stable (79-89% of initial). Given this result, the EC50 values were expressed in terms of the average exposure concentrations that were calculated to be: 0.11, 0.42, 1.0, 2.4 and 5.4 mg/L.

At average exposure concentrations of 0 (control), 0.11, 0.42, 1.0, 2.4 and 5.4 mg/L, respectively, immobility was 0%, 0%, 0%, 5%, 100% and 100% after 24 hours, and 0%, 0%, 0%, 95%, 100% and 100% after 48 hours. The 48h-EC50 was 0.73 mg/L (95% confidence interval between 0.63 and 0.82 mg/L).