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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2017 - 01 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd. 3 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Physical appearance: colourless to pale yellow liquid
Storage conditions: at room temperature protected from light, container flushed with nitrogen.
Specific details on test material used for the study:
- The test item is isoluble in water.
- Specific gravity/density: 1.059 (20ºC) and 1.057 (25ºC)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤ -15°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Direct application to test medium. The test solution was prepared at a loading rate of 100 mg/L applying a 3-day period of stirring to ensure maximum dissolution of the test item in medium. The obtained aqueous mixture was allowed to settle for a period of one hour. Thereafter, the clear and colourless Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg CaCO3/L
Test temperature:
19-20 °C (throughout the test)
pH:
7.9-8.1 (throughout the test)
Dissolved oxygen:
At the start of the test: 9.6-10 mg O2/L
At the end of the test: 8.8-9.0 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: 1.0, 2.2, 4.6, 10, 22, 46 and 100% of a saturated solution prepared at a loading rate of 100 mg/L
Measured concentrations: at t=0: 0.906, 2.01, 4.05, 9.09, 20.1, 42.0 and 91.4 mg/L; at t=48: 0.840, 1.91, 3.94, 8.79, 19.3, 40.6 and 88.9 mg/L, respectively to above stated nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 ml, all-glass
- Fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning, after 24 hours of exposure and at the end of the test, for all concentrations and the control. Temperature of the medium: continuously in a temperature control vessel.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100% of a saturated solution (SS) prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, after 48 hours of exposure, all daphnids became immobile in the highest concentration, while 20% immobility occurred at a test concentration of 10% of a SS prepared at 100 mg/L. Therefore, the expected EC50 was between 10 and 100% of a SS at 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (September 2017)

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Analysis showed that test concentrations remained stable during the exposure concentrations: test concentrations after 48 hours were 93-97% of the initial measured concentrations at t=0 (see table 1). Therefore, effect parameters were based on initial measured concentrations.
- At the end of the exposure concentration, all daphnids in the three highest test solutions were immobile, while no daphnids were immobile in the lowest three test concentrations and 5 and 15% immobility occurred in the 4.1 mg/L and 9.1 mg/L, respectively (see table 2).
- Microscopic observations showed no test item attached to the daphnids.
- The pH and temperature during the study remained within the limits prescribed by the study plan.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Tested concentrations: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- 48h-EC50: 0.41 mg/L, 95%-confidence interval: 0.34-0.46 mg/L
- Other: results fell within the historical range.
Reported statistics and error estimates:
The EC50-values were calculated from the weibits of the percentages affected daphnids and the logarithms of the corresponding test item concentrations (initially measured) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1 Measured concentrations during the final test

Time of sampling [hours]

Date of sampling

Date of analysisa

Percentage of SSb [%]

Analyzed concentration [mg/L]

Relative to initial [%]

0

23 Oct 2017

01 Nov 2017

0

n.d.

 

 

 

 

1.0

0.906

 

 

 

 

2.2

2.01

 

 

 

 

4.6

4.05

 

 

 

 

10

9.09

 

 

 

 

22

20.1

 

 

 

 

46

42.0

 

 

 

 

100

91.4

 

48

25 Oct 2017

01 Nov 2017

0

n.d.

n.a.

 

 

 

1.0

0.840

93

 

 

 

2.2

1.91

95

 

 

 

4.6

3.94

97

 

 

 

10

8.79

97

 

 

 

22

19.3

96

 

 

 

46

40.6

97

 

 

 

100

88.9

97

a     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

b     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

n.d.Not detected.

n.a.Not applicable.

Table 2 Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Test item initial concentration (mg/L)

Control

0.91

2.0

4.1

9.1

20

42

91

0

A

5

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

20

 

 

24

A

0

0

0

0

1

5#

5

5

B

0

0

0

0

1

5

5

5

C

0

0

0

0

0

5

5

5

D

0

0

0

0

1

5

5

5

Total immobilised

0

0

0

0

3

20

20

20

Effect %

0

0

0

0

15

100

100

100

 

 

 

 

 

 

 

 

48

A

0

0

0

1

1

5

5

5

B

0

0

0

0

1

5

5

5

C

0

0

0

0

0

5

5

5

D

0

0

0

0

1

5

5

5

Total immobilised

0

0

0

1

3

20

20

20

Effect %

0

0

0

5

15

100

100

100

#Microscopic observation revealed no test item attached to the daphnids

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'overall remarks'
Conclusions:
In an acute toxicity study, performed according to OECD 202 and GLP principles, the 48-h EC50 of Florantone T towards Daphnia magna was determined to be 12 mg/L (based on initial concentrations).