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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:
november 2017 - April 2018
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Solvent N1
- Physical state: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Manufacturer / Batch 24/02/2016
- Expiration date of the lot/batch: 31 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a dry, well ventilated dark location at ambient temperature/10–30°C
- Stability under test conditions: Chemically stable. Due to the presumed high volatility of the test substanceitem, specific measures for avoiding volatilizsation were applied during all the phases of the experimental work.
- Solubility and stability of the test substance in the solvent/vehicle: Not Applicable / No solvent used.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not Applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Control and test solutions were sampled in duplicate.
The day-0 samples of the test solutions (for control and all the tested concentrations) were taken directly from the volumetric flasks, i.e. immediately after preparation of the solutions.
The 48-h samples were taken from the test vessels immediately after determination of the biological parameters, with special attention on quick sample transfer in order to minimise losses of the test item during sampling. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded (20 mL).
After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18°C.
Samples were transferred to the test site for chemical analysis under the required storage conditions. The storage conditions were established in non-GLP pre-tests. According to the results of such non-GLP pre-test the samples are demonstrated to be stable if maintained at a temperature of ≤ -18°C for a storage period of up to 10 weeks.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
As the test item is of limited water-solubility and assumed to be highly volatile, the preparation procedure was set taking into account aspects of OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment Number 23. ENV/JM/MONO(2000)6.

A saturated stock solution was prepared at a nominal loading rate of 100 mg/L by adding 77 µL of the test item to 1027 g of the test medium, resulting in a loading rate of 100 mg test item/L. This stock solution (S1) was slowly stirred (300 rpm) for 22 hours at ambient temperature in the dark with minimised headspace. Thereafter this stock solution was left to settle for half an hour. The stock solution was visually examined for undissolved/particulate matter in the free water column.
The clear free water column of the stock solution S1 was used immediately to prepare the desired test item concentrations using a volumetric pipette for removal of S1 solution which was transferred to the respective volumetric flasks (C1-C4) that had been pre-filled as far as operationally possible with an appropriate volume of test medium prior to addition of the required volume of S1.

The test vessels were filled with the respective test solutions as far as operationally possible to avoid headspace above the test solutions, air-tightly closed and placed onto the testing area under test conditions.
The steps involving transfer and dilution of stock solution to the test solutions until closing of the test vessels were performed as quickly as possible in order to minimise air contact for the solutions during the manipulation steps; between removals of stock and test solutions, the respective bottles were closed.

Subsequently, the daphnids (up to 5 daphnids at a time) were transferred to the designated test vessels containing fresh test solution. Immediately after adding the required number of daphnids, the test vessels were tightly closed; where necessary, headspace was minimised by adding test solution of the respective concentration level.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, Clone M10
- Age of parental stock (mean and range, SD): less than 35 days
- Age of the daphnids at the beginning of the test: less than 24 hours
- Feeding during test: no

HOLDING CONDITIONS
The culture conditions of Daphnia magna as summarised below are similar to the test conditions.

Material of stock vessel: glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 15 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Separation of adult and young daphnids: 2-5 times per week
Medium: Elendt medium M4
Renewal of medium: twice per week
Temperature: within required ranges (20 +- 2°C)
Photoperiod (light/dark) 16/8 h
Light intensity: within required ranges (50 to 1000 lx)
Aeration: none
Food: algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week

The selected holding vessels designated for production of the neonates for the test were cleared of all mixed-aged young daphnids on the day before test start. These vessels contained parental daphnids aged less than 35 days. On the day of test start (day 0) all newly produced neonates of these vessels were < 24 hours old. These neonates were collected for the test.

After temperature adaptation of the test solutions the daphnids were transferred into the test solutions by using an upside-down glass pipette. The test organisms were added to the test solutions in a randomised manner until all test vessels contained the required number of daphnids. The daphnids were exposed to the test item concentrations until the end of the test.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none

Test conditions

Hardness:
Water Hardness of the M4 medium were determined before use.
Water Hardness (°dH) = 14.2
Water Hardness (mgCaCO3) = 254 (Total hardness in mg/L CaCO3 was calculated by multiplication of the hardness-value in °dH by 17.86. ASV: Air saturation value)
Test temperature:
The temperature was measured and recorded in one replicate test vessel of the control and the highest test concentration at start of the test, after temperature adaptation of the test solutions, and at the end of the test.
Temperature: 18 to 22°C, constant within a range of 2°C
Actual measured: min = 19.2°C, max = 21.5°C
pH:
The pH was measured and recorded in one replicate test vessel of the control and the highest test concentration at start of the test, after temperature adaptation of the test solutions, and at the end of the test.
Actual measured: min = 7.3, max = 7.6
Dissolved oxygen:
The dissolved Oxygen was measured and recorded in one replicate test vessel of the control and the highest test concentration at start of the test, after temperature adaptation of the test solutions, and at the end of the test.
Actual measured: min = 7.5 mg/L, max = 8.8 mg/L
Salinity:
//
Conductivity:
Conductivity of the M4 medium were determined before use.
Conductivity (µS/cm) = 657
Nominal and measured concentrations:
Nominal: 100%, 57.1%, 32.7%, 18.7%, 10.7% of the Saturated Solution (SS) prepared at the loading rate of 100 mg/L.
Measured (expressed as geometric mean measured concentration over 48 h) : 4.60, 2.86, 1.58, 0.94, 0.45 mg/L.
Details on test conditions:
TEST SYSTEM
Due to the high volatility of the test item, specific measures for avoiding volatilisation were applied during all the phases of the experimental work.

- Type (delete if not applicable): Closed
- Test vessel: 60 mL glass beakers, air-tightly closed by screwcaps with PTFE lining with minimised headspace
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable / No vehicle used

TEST MEDIUM: Elendt medium M4
- Date of prepration: 09 November 2017
- Alkalinity: 1.0 HCO3
- Conductivity: 657 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: At start of the test after temperature adaptation of the test solutions, and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 50 to 1000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility at 24 and 48h. Sublethal effects recorded.

Chemical analysis of test concentrations: At start and at end of exposure.
Data evaluation: Evaluation of immobility. Probit analysis, Qualitative trend analysis by contrasts, Fisher exact test.

RANGE-FINDING STUDY
- Test concentrations:
Nominal: 100, 20, 4% of a Saturated Solution prepared at a loading rate of 100 mg/L; 100% of the aqueous fraction at a loading rate of 100 mg/L, taken at start: 5.08 mg/L
Measured in the 100% SS: at start: 5.08 mg/L, at the end (48h): 2.76 mg/L
- Results used to determine the conditions for the definitive study: All Daphnids immobile at the undiluted aqueous fraction at a loading rate of 100 mg/L. All Daphnids mobile at 20 and 4% of the aqueous fraction at a loading rate of 100 mg/L, and in the controls.
Reference substance (positive control):
not required
Remarks:
The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7). Most recent reference test with K2Cr2O7 (separate study): EC50(24 h) = 1.29 mg/L.

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
4.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Corresponding to 80.13 % of Saturated Solution prepared at 100 mg/L loading rate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 4.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: EC50 higher than the highest tested nominal concentration (100% of a saturated solution at a loading rate of 100 mg test item/L), corresponding to a mean measured concentration 4.60 mg test item/L.
Details on results:
A weak concentration-response relationship was observed. A maximum of 15% immobility was found at the highest tested concentration.

Sublethal observations on the mobile daphnids were clearly concentration-related. Daphnids showing reduced swimming activity were observed in all vessels at the highest treatments of 100% of saturated solution after 24 h. After 48 h mobile daphnids showed reduced swimming activity in only one test vessel at 100% of saturated solution. In addition, one mobile daphnids lay on the bottom of a test vessel at 57.1% of saturated solution after 48 h.

Statistical Analysis
- Qualitative trend analysis by contrasts showed that the linear trend was not statistically significant (p > 0.05)
- Fisher`s Exact Binomial Test with Bonferroni Correction resulted in no statistically significant effects at up to and including the highest concentration, therefore the NOEC for the test was set equal to 100% of the saturated solution at a loading of 100 mg/L (corresponding to the measured concentration of 4.60 mg/L (geom mean).
Results with reference substance (positive control):
Positive control not required.
The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7). Most recent reference test with K2Cr2O7 (separate study): EC50(24 h) = 1.29 mg/L.
Reported statistics and error estimates:
Probit Analysis
No statistically significant concentration-response relationship was found (p(F) > 0.05; i.e. the slope of the relationship is not significantly different from zero). Due to the lacking concentration/response the shown ECx may be not reliable.

Results of the Probit analysis: Selected effective concentrations (ECx) of the test item and their 95%-confidence limits (according to Fieller`s theorem):
Parameter EC10 EC20 EC50
Value [mg/L] 4.04 n.d. n.d.
lower 95%-cl n.d. n.d. n.d.
upper 95%-cl n.d. n.d. n.d.
n.d.: not determined

Slope function after Litchfield and Wilcoxon: 17.778.


NOEC DETERMINATION
The linear trend is not significant (p > 0.05); (Qualitative Trend Analysis by Contrasts)

Fisher`s Exact Binomial Test with Bonferroni Correction: Pair-wise comparisons between treatment and control on the multiple significance level (alpha is 0.05; one-sided greater). Pair-wise comparisons are performed sequentially using the adjusted Alpha* (= alpha/(k-1); k: number of comparisons (after Holm 1979)); Ho (no effect) is accepted, if the probability p > Alpha*.

Treatm.[%]** Introduced Mobile Immobile % Immobility p alpha* sign.
Control 20 20 0 0.0
10.7 20 19 1 5.0 0.500 0.025 -
18.7 20 20 0 0.0 1.000 0.050 -
32.7 20 19 1 5.0 0.500 0.017 -
57.1 20 19 1 5.0 0.500 0.013 -
100 20 17 3 15.0 0.115 0.010 -

** % of saturated solution at a loading rate of 100 mg/L; +: significant; -: non-significant

As there were no significant effects at up to and including the highest concentration, the NOEC of this test is 100% of the saturated solution at a loading of 100 mg/L.



Any other information on results incl. tables

All validity criteria were fulfilled as required by the test guideline:

 

Required:

Found:

immobilised daphnids in the control: <=10%

0%

dissolved oxygen concentration in control and test vessels
at the end of the test: >=
3 mg/L

>= 7.5mg/L

 Signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water, did not exceed 10 per cent of the daphnids in the controls (actually observed: 0%).

-----------------------------------------

Observations concerning the behaviour of the mobile daphnids (number of daphnids showing the effect described below).

nominal concentration
[% of saturated solution]

replicate

control

10.7

18.7

32.7

57.1

100

introduced:

 

20

20

20

20

20

20

24 h

A

0

0

0

0

0

4c

 

B

0

0

0

0

0

5c

 

C

0

0

0

0

0

3c

 

D

0

0

0

0

0

5c

mobile:

 

20

19

20

19

19

18

48 h

A

0

0

0

0

0

3c

 

B

0

0

0

0

0

0

 

C

0

0

0

0

0

0

 

D

0

0

0

0

1b

0

mobile:

 

20

19

20

19

19

17

Explanations:

              0            No adverse observations compared to the controls; no special observations in the controls.

              b            Daphnids lying on the bottom of the test vessel

              c            Daphnids showed reduced swimming activity compared to the controls

              -             No surviving daphnids

----------------------------------------

Summary of measured concentrations during the test.

nominal concentration
[% of saturated solution]1

test period [h]

age of test solution [h]

measured concentration [mg test item/L]

 

geometric mean measured concentration over 48 h [mg test item/L]

0 (control)

0

0

n.d.

 

n.a.

10.7

0

0

0.43

 

n.a.

18.7

0

0

1.10

 

n.a.

32.7

0

0

1.80

 

n.a.

57.1

0

0

2.89

 

n.a.

100

0

0

5.72

 

n.a.

0 (control)

48

48

n.d.

 

n.d.

10.7

48

48

0.46

 

0.445

18.7

48

48

0.81

 

0.944

32.7

48

48

1.38

 

1.58

57.1

48

48

2.84

 

2.86

100

48

48

3.70

 

4.60

1% of saturated solution at a loading rate of 100 mg/L;Limit of Quantification (LOQ): 0.05 mg/L; n.d.: < 0.015 mg/L (< 30% of LOQ)

The observed decrease of measured test concentrations (C2, C3, C4, C5) from 0 h to 48 h may be explained by the presumed volatility of the test item from water.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
A weak concentration-response relationship for Mobility was observed. A maximum of 15% immobility was found at the highest tested concentration.
The EC50(48 h) was therefore higher than the highest tested nominal concentration (100% ofa saturated solution at a loading rate of 100 mg test item/L), corresponding to a mean measured concentration 4.60 mg test item/L.
Executive summary:

The aim of the study wasto determine the acute toxicity of the test item to the water fleaDaphnia magna. The study was conducted according to OECD test guideline No. 202 and GLP compliant.

A series of concentrations of the test item in aqueous solution was prepared. The test organisms were exposed to these treatments, as well as to controls without the test item, for a period of 48 hours under static test conditions.Those animals not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile.

The data obtained were analysed in order to estimate the concentration that would cause a x%immobilisation, i.e. ECx (e.g. EC50, EC20, EC10), and, where possible, a No-observed Effect concentration (NOEC).

As the test item is of limited water-solubility and assumed to be highly volatile, the preparation procedure of test solutions was set taking into account aspects of OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. 

 

To verify the nominally applied concentrations, samples were taken from all the test solutions and analytically measured. The samples designated for chemical analysis were shipped to the analytical test site and analysed under the responsibility of the principal investigator for analysis.

In a first pre-screening step, the stability of the aqueous samples at different storage conditions was tested in order to determine the appropriate storage conditions. The stability of stored samples over long-term period was evaluated and found appropriate.

 

 

Due to the presumed high volatility of the test item, specific measures for avoiding volatilisation (e.g. closed test vessels with minimised headspace) were applied during all the phases of the experimental work. 

The concentrations of the test item measured during the test were relatively stable, but not within 80-120% of the measured initial concentrations. Therefore the results were expressed as geometric mean measured concentrations over the 48 hours. The observed decrease of measured test concentrations from 0 h to 48 h may be explained by the presumed volatility of the test item from water.

A weak concentration-response relationship was observed. A maximum of 15% immobility was found at the highest tested concentration. Sublethal effects were noted on the mobile daphnids at the highest tested concentration (100% of saturated solution). Such effects were clearly concentration-related and partially recovered by the end of the test. In addition, one mobile daphnids lay on the bottom of a test vessel at 57.1% of saturated solution after 48 h.

The EC50(48 h)was therefore higher than the highest tested nominal concentration (100% ofa saturated solution at a loading rate of 100 mg test item/L), corresponding to a mean measured concentration 4.60 mg test item/L.

Allvalidity criteria were fulfilled as required by the test guideline.