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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:
28-Oct-2016 to 17-Nov-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13-April-2004
Deviations:
no
Principles of method if other than guideline:
Based on Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000, as the substance is an UVCB the test item preperations for testing were prepared using the water accomated fraction (WAF) approach.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and 100 mg/L
- Sampling method: For sampling 4.0 mL from the approximate centre of the test vessels were taken at 0 and 48 hours. At the end of each exposure period the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15°C) until analysis; stability at this storge temperature has been previously confirmed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accomadated fractions at 1.0, 10 and 100 mg/L were prepared seperately in medium with no solvent. The WAFs were prepared by adding known amounts of test item to the medium and allowing it to equilibrate over 3 days, stirring continuously. Following cessation of stirring and one hour of settling the aqueous phase was siphoned off for testing.
- Eluate: N/A
- Differential loading: N/A
- Controls: Test medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None, dissolved material was checked for using Tyndall's effect and no undissolved material was found.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: N/A (In-house culture)
- Justification for species other than prescribed by test guideline: N/A (guideline species)
- Source: In-house laboratory culture with a known history
- Age of parental stock (mean and range, SD): 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. Parental daphnids were more than two weeks old.
- Feeding during test: None
- Food type: N/A
- Amount: N/A
- Frequency: N/A

ACCLIMATION
- Acclimation period: N/A
- Acclimation conditions (same as test or not): ISO mdeium is used instead of Elendt M7 medium
- Type and amount of food: N/A
- Feeding frequency: N/A
- Health during acclimation (any mortality observed): N/A

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
N/A
Post exposure observation period:
N/A
Hardness:
180 mg/L CaCO3
Test temperature:
19-21 °C
pH:
8.0
Dissolved oxygen:
8.6-8.9 mg/L
Salinity:
N/A
Conductivity:
No data
Nominal and measured concentrations:
Concentraions were based as a loading rate and were 1.0, 10 and 100 mg/L. The measured concentraion at 100 mg/L was 1.93 mg/L at 0 hours and 1.81 mg/L (94 % of initial). The endpoints were represented as loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all glass
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 80 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: 5 for the controls and 100 mg/L loading rate, and 10 daphnids per vessel for the 1.0 and 10 mg/L loading rates
- No. of vessels per concentration (replicates): 2 for 1.0 mg/L and 10 mg/L loading rates and 4 for 100 mg/L loading rate
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts:

CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: 180 mg/L CaCO3
- Conductivity: Not reported
- Salinity: N/A
- Culture medium different from test medium:

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts:

CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach
the following concentrations:

Trace elements:
B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025
Se 0.0010
V 0.0003
Na2EDTA.2H2O 2.5 mg/L

Macro nutrients:
Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins:
Thiamine 75 μg/L
B12 1.0 μg/L
Biotin 0.75 μg/L

- Intervals of water quality measurement: 0 and 48 hours

OTHER TEST CONDITIONS
- Adjustment of pH: N/A
- Photoperiod: 16 hours
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobilisation at 24 and 48 hours

VEHICLE CONTROL PERFORMED: N/A

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L (Combined limit/range-finder)
- Results used to determine the conditions for the definitive study: No definitive study required due to lack of effect.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate
Remarks:
Loading rate of water accomadated fraction
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: 2 surface trapped daphnids at 100 mg/L at 24 hours
- Observations on body length and weight: N/A
- Other biological observations: N/A
- Mortality of control: 2 (10 %)
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: Uknown
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels:
The 24h-EL50 was 0.91 mg/L with a 95% confidence interval ranging from 0.77 to 1.1 mg/L.
The 48h-EL50 was 0.58 mg/L with a 95% confidence interval ranging from 0.49 to 0.67 mg/L.
- Limit test: No
- Dose-response test: Yes
- ECx: see above
- Other:
Reported statistics and error estimates:
The 48h-EL50 was >100 mg/L. No statistical analysis is needed as no/minimal effects were seen.

Table1. Number of introduced daphnids and incidence of immobility

Time (h)

Replicate

Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol,cyclized
Loading rate (mg/L)

Control

1.0

10

100

0

A

5

5

5

5

B

5

5

5

5

C

5

5

D

5

5

Total introduced

20

10

10

20

24

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

2

 

 

0 [2]

Total immobilised

2

0

0

0

Effect %

10

0

0

0

 

 

 

 

48

A

0

0

0

0

B

0

0

0

1

C

0

 

 

0

D

2

 

 

0

Total immobilised

0

0

0

1

Effect %

10

0

0

5

[ ]: number of daphnids observed trapped at the surface of the testsolutions[BL1] [KP2] .These organisms were reimmersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:
yes
Remarks:
Validity criteria as stipulated by OECD 202 were fulfilled
Conclusions:
The 48h-EL50 was >100 mg/L based on nominal concentration loading rates into the test system as water accommodated fractions.
Executive summary:

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

The batch of reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2- methylenedecahydro-1-naphthalenyl]-1-penten-3-ol,cyclized tested was an off white paste and a UVCB. The test item was not completely soluble in test medium at the loading rates initially prepared.

All test concentrations were prepared separately as water accommodated fractions (WAFs) due to the UVCB nature of the test item. Measured amounts of the test item were added directly to test medium and mixed for three days to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in the aqueous phase. Following cessation of mixing and a one hour period of settling (to allow phase separation) the aqueous phase, i.e. the WAF, was siphoned off for testing. Just before testing the WAFs were checked for tyndall effect, using a Laser pen.

A combined limit/range-finding test was performed exposing twenty daphnids per group (four replicates, five daphnids per replicate) to a control and a WAF at 100 mg/L. In addition, ten daphnids per group (two replicates, five daphnids per replicate) were exposed to WAFs at 1.0 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

The effect parameters were based on the initial loading rate instead of measured concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48h-EL50 was >100 mg/L based on nominal loading rates into the test system as water accommodated fractions.

Description of key information

48 h EL50 (Daphnia magna) = >100 mg/L, OECD 202 Daphnia sp. acute immobilisation test, (Anon., 2017)

Key value for chemical safety assessment

Additional information

The 48h-EL50 was >100 mg/L based on nominal concentration loading rates into the test system as water accommodated fractions.