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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:
16 Jan - 13 Apr 2017
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
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test item concentrations
- Sampling method: Samples were taken at test start and test end
- Sample storage conditions before analysis: Samples were stored at 2 to 8 °C until they were analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading:
The test item was weighed in ELENDT M4 dilution water followed by shaking on a shaking machine for 24 h being necessary to dissolve the test item sufficiently in the test medium. Thereafter the suspensions were passed over glass fibre filter and membrane filter in order to separate the non dissolved fraction from the dissolved fraction. The clear eluates were used directly without any further dilution steps.
- Controls: Yes
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: STRAUS, clone 5
- Source: Originally derived from the lnstitut National de Recherche Chimique Appliquee, France in 1978
- Age of parental stock (mean and range, SD): Not older than 24 h
- Feeding during test: No

ACCLIMATION
- Acclimation period: Cultivation of the daphnia is performed in a way that the animals are transferred in new test medium at an interval of 1 to 3 days.
- Acclimation conditions (same as test or not): Same
- Type and amount of food: Desmodesmus subspicatus- and/or Chlorella-cells
- Feeding frequency: An interval of 1 to 3 days

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Daphnias used for the test are not older than 24 h. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 h.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.7 - 20.9 °C (Control)
20.8 - 20.9 °C (Test item concentrations)
pH:
8.89 - 9.06 (Control)
8.95 - 9.22 (Test item concentrations)
Dissolved oxygen:
7.8 - 8.2 mg O2/L (Control)
7.4 - 8.2 mg O2/L (Test item concentrations)
Nominal and measured concentrations:
Control, 11.1, 22.2, 44.4, 88.8 and 111.1 mg test item/L (nominal)
Control, 9.99, 20.0, 40.0, 88.8 and 100 mg active substance/L (nominal)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: Open
- Material, size, headspace, fill volume: 100 mL glass carboys
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt medium M4 were used to prepare the test solutions.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Test start and test end

OTHER TEST CONDITIONS
- Photoperiod: 6 h of light and 8 h of darkness.

EFFECT PARAMETERS MEASURED: Mobility of the daphnia; examined by the determination of the swimming behavior of the daphnids after gentle agitation.

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations: 1.0; 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the screening test the test item concentration range of the main test was performed.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
80.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
22.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
44.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL10
Effect conc.:
12.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EL20
Effect conc.:
24.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
72.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL10
Effect conc.:
11.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EL20
Effect conc.:
21.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (dissolved fraction)
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Mortality of control: no
Results with reference substance (positive control):
The reference substance was tested twice a year. The value of the EC50 (24 h) was at 0.95 mg/L potassium dichromate. This value is in accordance with the recommendations of 0.6 to 2.1 mg/L K2Cr2O7 given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test)
Reported statistics and error estimates:
Statistical evalaution was done using the commercial computer program ToxRat Professional Version 3.2.1

Biological Results:

Table 1: Ratio of immobilised Daphnids

Nominal Concentration of the test item

(mg a.i./L)

Ratio immobilised Daphnia [%]

24 h

48 h

Control

0 [0/20] 1)

0 [0/20] 1)

9.99

0 [0/20] 1)

0 [0/20] 1)

20.0

0 [0/20] 1)

5 [1/20] 1)

40.0

65 [13/20] 1)

75 [15/20] 1)

79.9

20 [4/20] 1)

95 [19/20] 1)

100.0

30 [6/20] 1)

100 [20/20] 1)

1) Numbers in brackets give the absolute numbers of immobilized animals at each concentration.

 Table 2: Endpoints based on Test item concentration

 

Basis: Nominal Concentrations [mg test item/L] ("WAF")

t24hr

t48hr

NOEL

22.2

22.2

LOEL

44.4

44.4

EL10

18.9

12.8

EL20

40.3

24.1

EL50

171.1

80.8

 Table 3: Endpoints based on active ingredient

 

Basis: Nominal Concentrations of Active Substance ("WAF")

[mg Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato- kS)bis(cyclohexanamine)-,(T-4)- /L]

t24hr

48hr

NOEL

20.0

20.0

LOEL

40.0

40.0

EL10

17.0

11.5

EL20

36.3

21.7

EL50

154.0

72.7

   

Analtical Results:

As a conclusion of the analytical part of this study, it can be stated that the test item concentrations remained sufficiently stable during incubation of 48 h. As a result of the supporting analyse it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%.

Formula of the active Substance: C24H5 4N20 4P2S2Zn

According to this formula the contents of the different elements in the molecule are distributed as:

C: 46.04%; H: 8.69%; N: 4.7%; O: 10.22%; P: 9.89%; S: 10.24%; Zn: 10.44%

  

Table 4: Basis P the following recovery rates are calculated:

 

 

Time

nom. Conc. of active Substance [mg/L]

Basis P

 

mg Test ltem/L

 

% Nominal Conc.

 

Recovery % of t0

t0

t0

t0

t0

t0

t0

Control

10

20

40

80

100

0

1.547

2.952

3.559

8.959

9.929

-

15.5

14.8

8.9

11.2

9.9

-

-

-

-

-

-

 

Time

nom. Conc. of active Substance [mg/Ll

Basis P

mg Test ltem/L

% Nominal Conc.

Recovery % of t0

t48h

Control

0

-

-

t48h

10

1.234

12.3

79.7

t48h

20

2.750

13.8

93.2

t48h

40

3.246

8.1

91.2

t48h

80

8.686

10.9

97.0

t48h

100

9.353

9.4

94.2

 Table 5: Basis S

 

Time

nom. Conc. of active Substance [mg/L]

Basis S

mg Test ltem/L

% Nominal Cone.

Recovery % of 10

t0

t0

t0

t0

t0

t0

Control

10

20

40

80

100

0

0.781

3.613

8.398

21.777

24.023

-

7.8

18.1

21.0

27.2

24.0

-

-

-

-

-

-

t48h

t48h

t48h

t48h

t48h

t48h

Control

10

20

40

80

100

0

3.906

7.324

8.301

22.070

20.996

-

39.1

36.6

20.8

27.6

21.0

-

500.0

202.7

98.8

101.3

87.4

 Table 6: Basis Zn

 

 

Time

 

nom. Conc. of active Substance [mg/L]

Basis Zn

 

Mean Recoveries,

% of 10, total [Basis P, S, Zn]

 

mg Test ltem/L

 

% Nominal Conc.

 

Recovery% of t0

t0

t0

t0

t0

t0

t0

Control

10

20

40

80

100

0

0.412

0.460

0.546

0.584

0.718

-

4.1

2.3

1.4

0.7

0.7

-

-

-

-

-

-

-

-

-

-

-

-

t48h

Control

0

-

-

-

t48h

10

0.632

6.3

153.5

244.4

t48h

20

0.766

3.8

166.7

154.2

t48h

40

0.843

2.1

154.4

114.8

t48h

80

1.561

2.0

267.2

155.2

t48h

100

1.236

1.2

172.0

117.9

Table 7: Validity criteria for OECD 202.

Criterion from the guideline

Outcome

Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

No mortality was observed

Yes

The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

oxygen concentrations was above 60% of the saturated value (ca 5 mg O2/L)

Yes

 

Validity criteria fulfilled:
yes
Conclusions:
The EL50 was determined to be at 80.8 mg/L after 48 h and the NOEL was observed to be at 22.2 mg/L .

Description of key information

EL50 (48 h) = 80.8 mg test item/L (Daphnia magna, nominal, OECD 202, key study)

EL50 (48 h) = 72.7 mg active ingredient/L (Daphnia magna, nominal, OECD 202, key study)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
72.7 mg/L

Additional information

One experimental sutdy on the short-term toxicity of the test item to the freshwater species Daphnia magna is available (Lebertz, 2017b). The study was performed according to OECD guideline 202. The nominal concentrations were verified by analytical monitoring. All studies were performed according to GLP. Due to the moderate water solubility the test item was weighed in test medium followed by shaking on a shaking machine for 24 h and passed over glass fibre filter and membrane filter. The clear eluates were used directly without any further dilution steps. Daphnia magna was exposed over a period of 48 h without feeding in a static system to nominal concentrations of 11.1, 22.2, 44.4, 88.8 and 111.1 mg test item/L (nominal). Based on the active ingredient D. magna was exposed to the concentrations of 9.99, 20.0, 40.0, 88.8 and 100 mg active substance/L (nominal). Under the conditions used for the test, there was an immobilisation of D.magna at concentrations ≥22 mg test item/L representing 20 mg/L of active substance/L. As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation of 48 h. As a result of the supporting analyse it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%. The EL50 was determined to be at 80.8 mg test item/L after 48 h and the NOEL was observed to be at 22.2 mg test item/L. Based on the active ingredient the EL50 was determined to be at 72.7 mg a.i./L and the NOEL was observed to be at 20.0 mg a.i./L.