Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2018 to 13 April 2018
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
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the corresponding old or expired pooled replicates (R1 to R4) at 24 and 48 hours for quantitative analysis.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. A set of duplicate samples was taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Due to the low aqueous solubility and complex nature of the test material, for the purposes of the study the test medium was prepared as a WAF of the test material.
- Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test material in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in ISO media and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon. Visual examination of the WAFs showed that undissolved test material was dispersed throughout the water column. As such the aqueous phase was removed by filtering through a glass wool plug (2 to 4 cm in length) and one sheet of filter paper. After filtration no undissolved test material was observed to remain in the media. Samples were taken for chemical analysis.
- It was evident from this work that increasing the stirring period significantly increased the amount of Samarium, Europium and Gadolinium in the WAF between the 24-Hour and 96-Hour preparation periods and so preparation of the WAF was a 95-Hour stirring period followed by a 1-Hour standing period.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: In-house laboratory cultures.
- Age: Less than 24 hours old.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
- Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old (1st instar). These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3.
Test temperature:
20 - 23 °C
pH:
7.9 - 8.1
Dissolved oxygen:
8.4 - 9.0 mg O2/L
Nominal and measured concentrations:
Nominal Loading Rates: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass jars
- Type: The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: Approximately 100 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): Semi-static test conditions were employed in the test in an effort to maintain dissolved test material concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- An initial test was conducted at 10, 18, 32, 56 and 100 mg/L, however, it appeared that the 56 and 100 mg/L loading rates had been dosed incorrectly, as such, the test was repeated. Nominal amounts of test material (50, 90, 160, 280 and 500 mg) were each separately added to the surface of 5 litres of test water to give the 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test material, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. As a precautionary measure, a glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test material to be visible.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water – ISO Medium: 294 mg/L CaCl2.2H2O, 123 mg/L MgSO4.7H2O, 65 mg/L NaHCO3 and 5.8 mg/L KCl. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
- Culture medium different from test medium: Yes. Culture medium: Reconstituted Water – Elendt M7 Medium: 0.715 mg/L H3BO3, 0.090 mg/L MnCl2.4H2O, 0.077 mg/L LiCl, 0.018 mg/L RbCl, 0.038 mg/L SrCl2.6H2O, 0.004 mg/L NaBr, 0.016 mg/L Na2MoO4.2H2O, 0.004 mg/L CuCl2.2H2O, 0.013 mg/L ZnCl2, 0.010 mg/L CoCl2.6H2O, 0.0033 mg/L KI, 0.0022 mg/L Na2SeO3, 0.00058 mg/L NH4VO3, 0.625 mg/L Na2EDTA.2H2O, 0.249 mg/L FeSO4.7H2O, 293.8 mg/L CaCl2.2H2O, 64.8 mg/L NaHCO3, 123.3 mg/L MgSO4.7H2O, 10 mg/L Na2SiO3.9H2O, 5.8 mg/L KCl, 0.274 mg/L NaNO3, 0.184 mg/L K2HPO4, 0.143 mg/L KH2PO4, 0.075 mg/L Thiamine hydrochloride, 0.0010 mg/L Cyanocobalamine (vitamin B12) and 0.00075 mg/L D(+) biotin (vitamin H).
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: Between 200 and 1 200 lux

EFFECT PARAMETERS MEASURED:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: Nominal loading rates of 1.0, 10 and 100 mg/L.
- Nominal amounts of test material (5, 50 and 500 mg) were each separately added to the surface of 5 litres of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test material, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1-Hour. Observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water columns and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that no micro-dispersions remained. In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised daphnids were recorded.
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
other: NOEL
Effect conc.:
100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
other: NOEL
Effect conc.:
100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST

- No immobilisation was observed at 1.0 and 10 mg/L loading rate WAF, however, 30 % immobilisation was observed at 100 mg/L loading rate WAF.
- A sub-lethal effect of exposure was observed in the 100 mg/L loading rate WAF test concentration. This response was reduced mobility.
- Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
- Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of 0.35 to 0.21 mg/L as Samarium, 0.0059 to 0.034 mg/L as Europium, 0.0091 and 0.050 mg/L as Gadolinium. There was a significant decline in the measured concentrations at 48 hours indicating that the test material was not stable under test conditions.


DEFINITIVE TEST

Chemical Analysis of Test Loading Rates
- Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.10 mg/L as Samarium, 0.0025 to 0.017 mg/L as Europium and 0.0056 to 0.029 mg/L as Gadolinium. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.013 to 0.098 mg/L as Samarium, 0.0024 to 0.018 mg/L as Europium and 0.0054 to 0.037 mg/L as Gadolinium.
- The dissolved test material may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Immobilisation Data
- There was no significant immobilisation in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. The NOEL after 24 and 48 hours exposure was 100 mg/L loading rate WAF.

Sub-Lethal Effects
- A sub-lethal effect of exposure was observed in the 100 mg/L loading rate WAF. This response was reduced mobility.

Validation Criteria
- The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
- Temperature was maintained at 20 °C to 23 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- Throughout the test the light intensity was observed to be in the range 691 to 821 lux.

Vortex Depth Measurements
- The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Material Solubility
- Observations on the test media were carried out during the mixing and testing of the WAFs.
- At the start and end of the mixing period the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be clear colourless water columns with test material particles floating on the surface and dispersed throughout. After 1-Hour standing period all loading rates were observed to remain as at the start of stirring. Microscopic examination of the WAFs showed no micro-dispersions to be present however, as a precautionary measure, the WAFs were removed by filtering through a glass wool plug (2 to 4 cm in length). Microscopic examination after filtering showed no micro-dispersions to be present. During the test the control and all test solutions were observed to be clear, colourless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes. The results from the positive control with potassium dichromate were within the normal range for this reference item.
- Test concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
- Exposure conditions for the positive control were similar to those in the definitive test.
- Analysis of the immobilisation data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:
- 24 h: EC50 = 0.61 mg/L, NOEC = 0.32 mg/L and LOEC = 0.56 mg/L
- 48 h: EC50 = 0.54 mg/L, NOEC = 0.32 mg/L and LOEC = 0.56 mg/L
The No Observed Effect Concentration is based upon equal to or less than 10 % immobilisation at this concentration.
Reported statistics and error estimates:
An estimate of the EL50 values was given by inspection of the immobilisation data.

Table 1: Analytical Results for Test Samples

Time Point

[Hours]

Nominal Loading Rate

cnom

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Samarium in Test Sample

c

[mg/LSm]

Determined Concentration of Europium in Test Sample

c

[mg/LEu]

Determined Concentration of Gadolinium in Test Sample

c

[mg/LGd]

0

(Fresh)

Control

1.02

<LOQ

<LOQ

<LOQ

10

1.02

0.0131

0.00247

0.00563

18

1.02

0.0159

0.00318

0.00798

32

1.02

0.0167

0.00346

0.00917

56

1.02

0.0172

0.00367

0.0105

100

1.02

0.0168

0.00379

0.0117

24

(Old)

Control

1.02

<LOQ

<LOQ

<LOQ

10

1.02

0.0126

0.00239

0.00540

18

1.02

0.0147

0.00294

0.00739

32

1.02

0.0149

0.00313

0.00837

56

1.02

0.0156

0.00342

0.0100

100

1.02

0.0153

0.00348

0.0111

24

(Fresh)

Control

1.02

<LOQ

<LOQ

<LOQ

10

1.02

0.0847

0.0136

0.0206

18

1.02

0.0316

0.00549

0.0102

32

1.02

0.0366

0.00637

0.0122

56

1.02

0.0278

0.00520

0.0122

100

1.02

0.103

0.0170

0.0288

48

(Old)

Control

1.02

<LOQ

<LOQ

<LOQ

10

1.02

0.0379

0.00635

0.0107

18

1.02

0.0228

0.00414

0.00862

32

1.02

0.0269

0.00504

0.0114

56

1.02

0.0262

0.00523

0.0139

100

1.02

0.0982

0.0175

0.0365

LOQ = Limit of Quantification

Table 2: Cumulative Immobilisation Data and Observations in the Range‑finding Test

Nominal Loading Rate
(mg/L)

Observations (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilised Daphnia

Observations

Cumulative Immobilised Daphnia

Observations

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Control

0

0

5N

5 N

0

0

5 N

5 N

1.0

0

0

5 N

5 N

0

0

5 N

5 N

10

0

0

5 N

5 N

0

0

5 N

5 N

100

0

1

5 N

4 N

1

2

4 N

2 N 1 R

Rep = Replicate

R = Reduced mobility

N = Normal

Table 3: Cumulative Immobilisation Data and Observations in the Definitive Test

Nominal Loading Rate
(mg/L)

24 Hours

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

Nominal Loading Rate
(mg/L)

48 Hours

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

2

0

0

0

2

10

3 N

5 N

5 N

5 N

100

1

0

1

0

2

10

4 R

5 R

4 R

5 R

Rep = Replicate

N = Normal

R = Reduced movement

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 48-Hour EL50 value was greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.
Executive summary:

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2., under GLP conditions.

The study was performed to assess the acute toxicity of the test material to Daphnia magna in an Acute Immobilisation Test.

Due to the low aqueous solubility and complex nature of the test material, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to WAFs of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 20 to 23 °C under semi-static test conditions. The number of immobilised daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.10 mg/L as Samarium, 0.0025 to 0.017 mg/L as Europium and 0.0056 to 0.029 mg/L as Gadolinium. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.013 to 0.098 mg/L as Samarium, 0.0024 to 0.018 mg/L as Europium and 0.0054 to 0.037 mg/L as Gadolinium. The dissolved test material may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L loading rate WAF.

Under the conditions of this study, the 48-Hour EL50 value was greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.

Description of key information

Under the conditions of this study, the 48-Hour EL50 value was greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Additional information

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2., under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the acute toxicity of the test material to Daphnia magna in an Acute Immobilisation Test.

Due to the low aqueous solubility and complex nature of the test material, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to WAFs of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 20 to 23 °C under semi-static test conditions. The number of immobilised daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.10 mg/L as Samarium, 0.0025 to 0.017 mg/L as Europium and 0.0056 to 0.029 mg/L as Gadolinium. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.013 to 0.098 mg/L as Samarium, 0.0024 to 0.018 mg/L as Europium and 0.0054 to 0.037 mg/L as Gadolinium. The dissolved test material may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L loading rate WAF.

Under the conditions of this study, the 48-Hour EL50 value was greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.