α-phenyl-1H-benzimidazole-2-methanol

Short-term toxicity to aquatic invertebrates

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:

02 July - 30 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 18, 32, 56 and 100 % v/v (prepared from 100 % v/v saturated solution)
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from pooled replicates at 48 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution was prepared by stirring an excess (100 mg/L) of test item in Elendt M7 for a period of 24 hours prior to removing any undissolved test item present by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item. A series of dilutions was made from this saturated solution to give stock solutions of 10, 18, 32, 56 % v/v saturated solution. The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
- Eluate: Elendt M7
- Controls: Elendt M7 (four replicates)
- 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.): No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (water flea)
- Strain/clone: -
- Justification for species other than prescribed by test guideline: -
- Source: In-house culture
- Age of parental stock (mean and range, SD): -
- Feeding during test: No
- Food type: -
- Amount: -
- Frequency: -

ACCLIMATION
- Acclimation period: No acclimation period. The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
- Acclimation conditions (same as test or not): -
- Type and amount of food: -
- Feeding frequency: -
- Health during acclimation (any mortality observed): -

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

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: 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. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and GM300 suspension. 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. 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

As prescribed by guideline

Hardness:

Not reported

Test temperature:

21-22 ºC

pH:

7.9-8.1

Dissolved oxygen:

8.7-9.0 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 % v/v saturated solution
Measured (0 h): 7, 13, 24, 41 and 70 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass; 150 mL capacity filled to 100 mL with test solution.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): -
- Renewal rate of test solution (frequency/flow rate): -
- 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): -
- Biomass loading rate: -

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: In-house preparation of Elent M7 using reconstituted fresh water and salt stocks. The pH of the media was adjusted to 7.9 ±0.3 and stored at 21 ºC.
- Total organic carbon: -
- Particulate matter: -
- Metals: -
- Pesticides: -
- Chlorine: -
- Alkalinity: -
- Ca/mg ratio: -
- Conductivity: -
- Salinity: -
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. 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: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: Between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Testing with potassium dichromate routinely undertaken at the Testing Facility.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

41 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 70 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: Not reported
- Other biological observations: None observed
- Mortality of control: 0 %
- Other adverse effects control: No
- Abnormal responses: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item at concentrations of 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 immobilization data was carried out using the Trimmed Spearman-Karber method at 24 hours and the Binomial Distribution method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations;

48 h EC50 = 0.79 (95 % CI: 0.73 - 0.86 mg/L)
48 h NOEC = 0.56

The NOEC is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Table 1       Immobilisation and observation data

 

0 h measured concentration (mg/L)	24 h
	Cumulative immobilised Daphnia (5 per rep.)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	5N
7.0	0	0	0	1	1	5	5N	5N	5N	5N
13	0	0	0	0	0	0	5N	5N	5N	5N
24	0	1	0	0	1	5	5N	4N	5N	5N
41	0	0	0	0	0	0	5N	5N	5N	5N
70	0	0	0	0	0	0	5N	5N	5N	5N
0 h measured concentration (mg/L)	48 h
	Cumulative immobilised Daphnia (5 per rep.)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	5N
7.0	0	0	0	1	1	5	5N	5N	5N	5N
13	0	0	0	0	0	0	5N	5N	5N	5N
24	0	1	0	0	1	5	5N	4N	5N	5N
41	0	5	3	1	9	45	5N	AI	2N	4N
70	5	4	5	5	19	95	AI	1N	AI	AI

N = normal

AI = All immobilised

 

Table 2       Water Quality Parameters

 

0 h measured concentration (mg/L)		0 h			24 h	48 h
		pH	mg O2/L	Temp ºC	Temp ºC	pH	mg O2/L	Temp ºC
Control	R1	7.9	8.9	22	21	7.9	9.0	21
7.0	R1	7.9	8.7	22	21	8.0	9.0	21
13	R1	8.0	8.7	22	21	8.0	8.9	21
24	R1	8.0	8.7	22	21	8.1	8.8	21
41	R1	8.0	8.8	22	21	8.1	8.9	21
70	R1	8.0	8.9	21	21	8.1	8.9	21

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50, based on immobility, for Daphnia magna under the conditions of this test was 41 mg/L.

Executive summary:

OECD 202 (2019) - The 48 h acute toxicity to Daphnia magna was studied under static conditions.  Test species were exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (four replicates flasks per concentration, each containing 5 daphnids) for 48 hours at a temperature of 21 - 22  ºC. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in Elendt M7 medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 7.0 to 70 mg/L. There was no significant change in the measured concentrations at 72 hours (7.0 to 71 mg/L) and so the results are based on the 0-Hour measured test concentrations only; 7.0, 13, 24, 41 and 70 mg/L.

Mortality/ immobilisation and sublethal effects were observed daily.

 

The 24 and 48 h EC₅₀ values were >70 and 41 mg/L, respectively.

  

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

 

 

Description of key information

48 h EC50: 41 mg/L; OECD 202; Squance, D. (2019)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

41 mg/L

Additional information

OECD 202 (2019) - The 48 h acute toxicity to Daphnia magna was studied under static conditions.  Test species were exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (four replicates flasks per concentration, each containing 5 daphnids) for 48 hours at a temperature of 21 - 22  ºC. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in Elendt M7 medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 7.0 to 70 mg/L. There was no significant change in the measured concentrations at 72 hours (7.0 to 71 mg/L) and so the results are based on the 0-Hour measured test concentrations only;7.0, 13, 24, 41 and 70 mg/L.

Mortality/ immobilisation and sublethal effects were observed daily.

 

The 48 h EC₅₀ was 41 mg/L.  The 48 h NOEC based on mortality/immobilization/sublethal adverse effects was 24 mg/L.

  

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.