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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:
18 July 2017 - 16 August 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:
13 April 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: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
13 March 2017
Specific details on test material used for the study:
Correction factor for purity: 2.63
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: at t=0 duplicate samples were taken from the freshly prepared test media, at t=48 duplicate test samples were taken from the test media by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: all samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test item was applied individually, directly to the test medium. Subsequently, the test solutions were stirred for 15 minutes.
- Controls: In the control, test water was used without addition of the test item.
- Evidence of undissolved material: the test item caused foaming in the test media of 54.4, 120 and 263 mg test item/L (20.7, 45.5 and 100 mg solids/L). Additionally, at a concentration of 263 mg test item/L (100 mg solids/L) the test medium was observed to be slightly turbid.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnia
- Clone: 5
- Source: in-house laboratory stock
- Age at study initiation: 1-21 hours old
- Age of parental stock: not indicated
- Feeding during breeding: daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of the test facility.
- Feeding during test: no

ACCLIMATION: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The daphnids were bred in the laboratories of the test facility under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg CaCO3/L
Test temperature:
19.9 to 20.5 °C at test start;
20.4 °C at test end
pH:
7.9 to 8.1 at test start;
7.9 at test end
Dissolved oxygen:
9.2 to 10.0 mg/L at test start;
8.6 to 8.8 mg/L at test end
Nominal and measured concentrations:
Nominal concentrations: 263, 120, 54.4, 24.7 and 11.3 mg test item/L (equivalent to 100, 45.5, 20.7, 9.4 and 4.3 mg solids/L)
Measured concentrations: see table 1 in 'any other information on results'
Mean measured concentrations were 79-97% of nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers of 100 mL, fill volume: 60 mL, covered with a lid
- 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: reconstituted water (Elendt ''M4'')
- Culture medium different from test medium: no
- Intervals of water quality measurement: water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 160 to 270 lux

EFFECT PARAMETERS MEASURED: The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, no immobility was observed in any of the concentrations tested. Therefore the EC50 was expected to exceed the highest tested concentration.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (January 2017)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
177 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
67.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solid fraction
Basis for effect:
mobility
Details on results:
- Abnormalities observed: no
- After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 120 mg test item/L (45.5 mg solids/L). At the highest test concentration of 263 mg test item/L (100 mg solids/L), 20 daphnids were immobile (see table 2).
- At test end, the test medium in the highest concentration of nominal 263 mg test item/L (100 mg solids/L) was observed to be slightly turbid.
- Analysis showed that the test concentrations remained 79-97% of nominal test concentrations. For the lowest concentration, the measured values at t=48 were below 80% of the nominal concentration, however, the nominal values were used for calculating the effect concentrations. This was done because the NOEC is 120 mg test item/L and the recoveries in the higher concentration range show nominal recoveries in the freshly prepared and aged test samples.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 48h-EC50: 1.06 mg/L
- Other: based on the 24h-EC50 of 1.48 mg/L, the sensitivity was according to the proposed OECD levels (24h-EC50 between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1 Measured concentrations

Sample description

Concentration found

D.F.

Concentration calculated

Concentration nominal

(mg test item/L)

Sampling day

Age (h)

 (mg test item/L)*

1

(mg test item/L)*

 (mg test item/L)

% of nominal*

Control

0

0

<LOD

1

n.a.

0.000

n.a.

Control

0

0

<LOD

1

n.a.

0.000

n.a.

Control

2

48

<LOD

1

n.a.

0.000

n.a.

Control

2

48

<LOD

1

n.a.

0.000

n.a.

11.3

0

0

10.153

1

10.153

11.300

90

11.3

0

0

9.954

1

9.954

11.300

88

11.3

2

48

7.761

1

7.761

11.300

69

11.3

2

48

7.645

1

7.645

11.300

68

 

 

 

 

 

 

Mean value (n=4):

79

 

 

 

 

 

 

RSD (n=4):

15

24.7

0

0

22.645

1

22.645

24.700

92

24.7

0

0

22.197

1

22.197

24.700

90

24.7

2

48

22.286

1

22.286

24.700

90

24.7

2

48

22.230

1

22.230

24.700

90

 

 

 

 

 

 

Mean value (n=4):

90

 

 

 

 

 

 

RSD (n=4):

1

54.4

0

0

51.625

1

51.625

54.400

95

54.4

0

0

53.194

1

53.194

54.400

98

54.4

2

48

51.910

1

51.910

54.400

95

54.4

2

48

52.049

1

52.049

54.400

96

 

 

 

 

 

 

Mean value (n=4):

96

 

 

 

 

 

 

RSD (n=4):

1

120

0

0

115.866

1

115.866

120.000

97

120

0

0

116.251

1

116.251

120.000

97

120

2

48

117.327

1

117.327

120.000

98

120

2

48

115.440

1

115.440

120.000

96

 

 

 

 

 

 

Mean value (n=4):

97

 

 

 

 

 

 

RSD (n=4):

1

263

0

0

110.633

2

221.266

263.000

84

263

0

0

116.767

2

233.534

263.000

89

263

2

48

113.672

2

227.345

263.000

86

263

2

48

107.259

2

214.517

263.000

82

 

 

 

 

 

 

Mean value (n=4):

85

 

 

 

 

 

 

RSD (n=4):

4

 

 

 

 

 

Overall mean value (n=20)

89

 

 

 

 

 

RSD (n=20)

10

The tabulated results represent rounded results calculated on the exact raw data

LOD: Limit of Detection = 2 mg test item/L

n.a.: not applicable; RSD: Relative Standard Deviation; D.F.: Dilution factor

italics: The values are below 80% of the nominal concentration, however, the nominal values were used for calculating the effect concentrations. This was done because the NOEC is 120 mg test item/L and the recoveries in the higher concentration range show nominal recoveries in the freshly prepared and aged test samples.

Table 2 Influence of Miranol BM Conc. on the mobility of Daphnia magna

Nominal concentration (mg test item/L)

No. of Daphnia tested

No. of immobilized Daphnia

% of immobilized Daphnia

 

 

After 24 h

After 48 h

After 24 h

After 48 h

Control

20

0

0

0

0

11.3

20

0

0

0

0

24.7

20

0

0

0

0

54.4

20

0

0

0

0

120

20

0

0

0

0

263

20

2

20

10           

100

Validity criteria fulfilled:
yes
Remarks:
See 'overall remarks' section
Conclusions:
The 48h-EC50 for Miranol BM Conc towards Daphnia magna was 177 mg/L (67.3 mg solids/L).
Executive summary:

A short term toxicity test was performed, according to OECD guideline 202 and GLP principles, to assess the potential of Miranol BM Conc to induce immobility in Daphnia magna. The test item was corrected for the solid fraction and added to the medium at nominal concentrations of 11.3, 24.7, 54.4, 120 and 263 mg test item/L (corresponding to 4.3, 9.4, 20.7, 45.5 and 100 mg solids/L). Twenty daphnia per concentration and a blank control, divided over four vessels, were exposed to the test solutions for 48 hours. The number of immobilised daphnids after 24 hours and 48 hours was recorded. Duplicate samples for analysis were taken at t=0 and t=48 to determine actual exposure concentrations. Analysis showed that the test concentrations remained 79 -97% of nominal test concentrations. The effect concentrations were based on nominal test concentrations since the measured concentration was below 80% of initial in the lowest concentration only and no effects were seen at this concentration.

Only in the highest test concentration an effect was observed. In the other test concentrations no daphnids became immobilised while in the highest test concentration 100% of the introduced daphnids became immobilised. The 48h-EC50 of Miranol BM Conc. was determined to be 177 mg/L (67.3 mg solids/L). All validity criteria were met and the study was considered to be valid.

Description of key information

Two studies with the registered substance are available, as well as 5 older studies with a substance analogue. The EC50 values for the registered substance are 67.4 mg solids/L and >100 mg solids/L. Comparing the older data with the results of the daphnia studies performed in 2017 show that the newer studies give a higher EC50 value. This is expected to be related to the degree of purity of the substances tested. The results from the acute studies with daphnids are considered more relevant for read across than the older reported EC50 values. See the read-across report in Section 13 for full details of the read-across rationale. The lowest EC50 value obtained with the registered substance is used as a worst-case key value for chemical safety assessment.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
67.3 mg/L

Additional information

The 48h-EC50 for Miranol BM Conc towards Daphnia magna was 177 mg/L (67.3 mg solids/L).