2-phenyl-1H-benzimidazole-5-sulphonic acid

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-04-28 to 2017-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

- Concentrations: all test concentrations and control
- Sampling method: Duplicate samples from the freshly prepared test medium at one treatment period of the first, second and last week of the test (Day 0, 7 and 16). Stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).


- Sample storage conditions before analysis: frozen (at about –20 °C) immediately after sampling

For each sampling time the concentration of the test item was determined in one of the duplicate test medium samples (freshly prepared test medium and aged samples with food), from the nominal concentration of 100 mg/L and the control. The analytical samples of the concentrations of 6.25 to 50 mg/L were not analyzed as these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

Vehicle:

no

Details on test solutions:

Prior to the start of the test and prior to each test medium renewal, the test medium of the highest nominal test concentration of 100 mg/L was freshly prepared by completely dissolving 200 mg of the test item (dosed in the range of 200.01 to 200.14 mg) in 2000 mL of test water using ultrasonic treatment for 15 minutes and intensive stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with adequate volumes of test water to prepare the lower concentrated test media.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Source: Daphnia Collection of the University of Basel, Switzerland
- Age of parental stock: at least 14 days old and not older than 4 weeks
- Feeding during test: yes
- Food type: food mixture - a suspension of green algae of the species Desmodesmus subspicatus (freshly grown) and a fish food suspension. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA GmbH, 49304 Melle, Germany) in 500 mL of M7 medium. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with M7 medium and boiled.
- Amount: 0.20 mg TOC per Daphnia per day
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (250 mg/L CaCO3)

Test temperature:

20 - 22 °C

pH:

7.6 - 8.8

Dissolved oxygen:

8.1 - 9.9 mg/L

Salinity:

NA

Conductivity:

none

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Measured concentrations: in the range of 98 - 100 % of the nominal concentrations for the 100 mg/L concentration. Tthe other concentrations were not analysed as it was not relevant for EC-calculation.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type: closed - covered with glass plates
- Fill volume: 80 mL
- Renewal rate of test solution: 48 or 72 hours renewal periods
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 Medium
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light, 8-hour dark cycle
- Light intensity: 19 - 20 μmol/m2s

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10, 100 mg/L

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, mortality and growth

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, mortality and growth

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, mortality and growth

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, mortality and growth

Details on results:

- The concentration of the test item measured in the test medium of the nominal test concentration of 100 mg/L were between 98 and 100 % of the nominal values.
- Survival of the test animals in the control and all test concentrations at the end of the test was 100 %.
- No immobilised test organisms were determined in the control and all test concentrations after 48 hours of exposure.
- The first young offspring released from their parent animals were recorded in the control and at all test concentrations at observation on Day 7. Thus, the time of the first brood was not affected by the test item up to and including the highest test concentration of 100 mg/L.
- The mean reproduction rate of the daphnids in the control was 159 ± 10 living offspring per surviving adult (mean ± standard deviation). No statistically significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the highest test concentration of 100 mg/L (Dunnett t-test, one-sided smaller, α = 0.05).
- The mean body length of the daphnids in the control was 4.75 ± 0.10 mm. At all test concentrations the mean body length was not significantly smaller than the control (Dunnett t-test, one-sided smaller, α = 0.05).
- The 21-day EC10 and EC50 values for the mortality, inhibition of the reproduction rate and body length could not be calculated, since no toxic effects of the test item on daphnia up to and including the highest test concentration of 100 mg/L was observed.

Range-Finding Test

Nominal Concentration [mg/L]	10 Day Mortality [%]	Mean Cumulative Offspring after 10 Days	Reduction of Cumulative Offspring [%]
Control 0.10 1.0 10 100	0 0 0 0 0	46 43 44 46 42	-- 6.4 3.3 -1.8 8.4

-- Not applicable

Validity

The test was considered to be valid since all relevant validity criteria have been fulfilled according to the OECD Guideline No. 211 (2012).

− Mortality of parent test animals in the control should not exceed 20 % at the end of the test. At the end of the test the mortality of the parent test animals was 0 %.

− The mean number of living offspring in the control should be at least 60 per surviving adult Daphnia at the end of the test. The mean numaber at the end of the test was 159.

− The coefficient of variation around the mean number of living offspring produced per parent animal in the control should be ≤25 %. In the control of the test it was 6.4 %.

Results

Table 1. Total Number of living young daphnids procedured by all adults (cumulative values)

Exposure day	Nominal Concentration of test item [mg/L]
	Control	6.25	12.5	25	50	100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21	0 0 0 0 0 0 0 51 134 134 260 564 564 606 977 977 977 992 1181 1181 1181 1587	0 0 0 0 0 0 0 65 151 151 319 583 583 668 985 985 985 1036 1195 1195 1195 1677	0 0 0 0 0 0 0 103 138 138 444 536 536 701 964 964 964 1065 1105 1105 1105 1559	0 0 0 0 0 0 0 83 151 151 391 570 570 699 973 973 1021 1146 1199 1248 1337 1597	0 0 0 0 0 0 0 42 146 146 308 570 570 707 982 982 982 1104 1183 1183 1270 1590	0 0 0 0 0 0 0 71 162 162 326 587 587 678 1003 1003 1003 1175 1225 1225 1364 1644
Number of Adults Surviving at Test End	10	10	10	10	10	10

Table 2. Body length [mm] of the surviving adults after 21 days of exposure

Replicate No.	Nominal Concentration of test item [mg/L]
	Control	6.25	12.5	25	50	100
0 1 2 3 4 5 6 7 8 9 10	4.84 4.52 4.71 4.84 4.71 4.71 4.84 4.77 4.71 4.84	4.90 4.90 4.77 4.77 4.65 4.58 4.84 4.84 4.77 4.71	4.65 4.65 4.84 4.84 4.84 4.65 4.77 4.77 4.77 4.77	4.77 4.65 4.77 4.71 4.65 4.71 4.45 4.58 4.77 4.65	4.52 4.45 4.71 4.65 4.84 4.65 4.71 4.84 4.65 4.65	4.77 4.52 4.77 4.65 4.77 4.45 4.65 4.71 4.65 4.58
Mean ± SD n	4.75 0.10 10	4.77 0.11 10	4.75 0.08 10	4.67 0.10 10	4.66 0.12 10	4.65 0.11 10
CV %	2.1	2.2	1.7	2.2	2.6	2.4
% of Control	-	101	100	98	98	98
STAT	-	n.s.	n.s.	n.s.	n.s.	n.s.

SD: Standard Deviation

CV %: Coefficinet of variation: (SD/mean) x 100 %

n.s.: Not statistically significantly lower than the control value according to a Dunnett's test, one-sided smaller α = 0.05

Analytic results

Table 3. Results for test samples

Sampling Day / Sample Age [d/h]	Nominal concentration of test item cnom [mg/L]	Measured concentration of test item x [mg/L]	Sample preparation factor F	Determined concentration of test item c [mg/L]	% of nominal concentration [%]
0/0	Control 100	n.d. 48.9	2 2	<LOQ 97.9	n.a. 98
2/48 (aged)	Control (a) 100 (a)	n.d. 50.1	2 2	< LOQ 100.2	n.a. 100
7/0	Control 100	n.d. 49.0	2 2	< LOQ 98.0	n.a. 98
9/48 (aged)	Control (a) 100	n.d. 49.5	2 2	< LOQ 99.0	n.a. 99
16/0	Control 100	n.d. 49.0	2 2	< LOQ 98.1	n.a. 98
19/72 (aged)	Control (a) 100 (a)	n.d. 49.9	2 2	< LOQ 99.8	n.a. 100

(a) = samples with feed

n.d. = no test item detected

n.a. = not applicable

LOQ: 32 mg/L

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, based on the results for survival, growth and reproduction of the test animals, the overall 21-day NOEC for the tets item was ≥100 mg/L.

Executive summary:

The long-term toxicity of the test item to aquatic invertebrates - Daphnia magna, was tested in a 21-day study performed according to OECD 211 and Regulation (EC) No. 2017/735 Method C.20. A semi-static test with 5 nominal concentrations (6.25, 12.5, 25, 50 and 100 mg/L) and a control was conducted. Per treatment and the control 10 daphnids were tested. Daphnids were individually exposed in glass beakers covered with glass plates to reduce evaporation losses containing 80 mL test medium. A test medium renewal was performed every 48 or 72 hours. The test concentration in the medium was determined with HPLC-UV. The measured concentrations were in the range of 99 to 100 % of the nominal concentrationof 100 mg/L, therefore, the biological results are based on the nominal concentration. The test item had no toxic effects on survival, growth and reproduction of Daphnia magna after the exposure period of 21 days up to and including the highest nominal test concentration of 100 mg/L. Thus, the 21-day NOEC of the test item was determined as ≥100 mg/L. The 21-day LOEC was above the highest test concentration of 100 mg/L. The 21-day EC10 and EC50 for the mortality, inhibition of the reproduction rate and body length of the daphnids were higher than 100 mg/L. Additionaly, the acute 48 -hour EC50 of immobility was determined and it was clearly higher than 100 mg/L.

Description of key information

In a semi-static 21 -days chronic toxicity test with Daphnia magna, the test item showed no toxic effects of reproduction, survival and immobilisation up to the maximum tested concentration of 100 mg/L. Therefore, the EC10 is >100 mg/L and the NOEC is determined to be ≥100 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

EC10 > 100 mg/L NOEC >= 100 mg/L

Effect concentration:

100 mg/L

Additional information

The long-term toxicity of the test item to aquatic invertebrates (Daphnia magna), was tested in a 21-day study performed according to OECD 211 and Regulation (EC) No. 2017/735 Method C.20. A semi-static test with 5 nominal concentrations (6.25, 12.5, 25, 50 and 100 mg/L) and control was conducted. Per treatment and the control 10 daphnids were tested. Daphnids were individually exposed in glass beakers covered with glass plates to reduce evaporation losses containing 80 mL test medium. A test medium renewal was performed every 48 or 72 hours. The test concentration in the medium was determined with HPLC-UV. The measured concentrations were in the range of 99 to 100 % of the nominal concentration, therefore, the biological results are based on the nominal concentration. The test item had no toxic effects on survival, growth and reproduction of Daphnia magna after the exposure period of 21 days up to and including the highest nominal test concentration of 100 mg/L. Thus, the 21-day NOEC of the test item was determined as ≥100 mg/L. The 21-day LOEC was above the highest test concentration of 100 mg/L. The 21-day EC10 for the mortality, inhibition of the reproduction rate and body length of the daphnids were higher than 100 mg/L. Additionally, the acute 48 -hour EC50 of immobility was determined to be > 100 mg/L.