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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from Jun. 04, 2012 to Nov. 23, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Determination of Algal Biomass: A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
Water Quality Criteria: The pH was measured and recorded in each treatment at the start and end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 50.05 mg of the test item completely in 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature in the dark. The pH of the test medium of the highest test concentration was adjusted from 4.4 to 6.5 with a 0.1 M sodium hydroxide solution. This adjusted solution was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Strain No.: 61.81 SAG
Supplier: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany)
The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
At the start of the test, 0.5 x 104 algal cells/mL were inoculated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L as CaCO3

Test temperature:

22 - 23 °C

pH:

test start: 7.1 - 7.4
test end: 8.3 - 8.5

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

nominal: 6.25, 12.5, 25, 50 and 100 mg/L
The measured concentrations of the test item in the test media of the test concentration of 100 mg/L were 101 % of the nominal values at the start and at the end of the test.

Details on test conditions:

Test Water: Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations.
Material: 50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.
Experimental Conditions: The test flasks were incubated in a temperature-controlled water bath at a temperature between 22 and 23 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 5400 Lux (range: 5150 to 5910 Lux, measured at nine places in the experimental area). The light intensity was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

The algal biomass in the samples was determined by measurement of the algal cell density using an electronic particle counter (Coulter Counter, Model Z2). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the control and from the test concentration of nominal 100 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

no data

Results with reference substance (positive control):

The result of the latest positive control test performed in February 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.97 mg/L (Harlan Study Number D50552), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.71 - 1.7 mg/L).

Reported statistics and error estimates:

Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.
The 72-hour EC10, EC20 and EC50 values of the test item could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test [Williams, 1971; Williams, 1972] or Welch t-test [Sachs, 1984], where appropriate.

Validity criteria fulfilled:

yes

Conclusions:

The biological results (based on nominal concentrations) of the study were as follows: 72-h EC50 (based on growth rate) >100 mg/L; 72-h LOEC (based on growth rate) >100 mg/L; 72-h NOEC (based on growth rate) ≥100 mg/L; 72-h EC50 (based on yield) >100 mg/L.

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3. The nominal concentrations of the test item of 6.25, 12.5, 25, 50 and 100 mg/L were tested in parallel with a control. The measured concentrations of the test item in the test media of the test concentration of 100 mg/L were 101 % of the nominal values at the start and at the end of the test. Thus, the correct dosing of the test item was confirmed. The test item was stable in the test media over the test period of 72 hours.

The biological results were related to the nominal concentrations of the test item. The biological results (based on nominal concentrations) of the study were as follows: 72-h EC50 (based on growth rate) >100 mg/L; 72-h LOEC (based on growth rate) >100 mg/L; 72-h NOEC (based on growth rate) ≥100 mg/L; 72-h EC50 (based on yield) >100 mg/L.

Description of key information

The toxicity to aquatic algae and cyanobacteria was determined in an OECD guideline study. The EC50 (growth rate) was determined to be > 100 mg/L. The NOEC was determined to be ≥100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The available test with algae was performed with the freshwater green algal species Pseudokirchneriella subcapitata according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3 in a 72-hour static test. The nominal concentrations of the test item of 6.25, 12.5, 25, 50 and 100 mg/L were tested in parallel with a control. The test item was stable in the test media over the test period of 72 hours.

The biological results were related to the nominal concentrations of the test item. The biological results (based on nominal concentrations) of the study were as follows: 72-h EC50 (based on growth rate) >100 mg/L; 72-h LOEC (based on growth rate) >100 mg/L; 72-h NOEC (based on growth rate) ≥100 mg/L; 72-h EC50 (based on yield) >100 mg/L.