Benzethonium chloride

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:

September - October 2001

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)

Specific details on test material used for the study:

TEST MATERIAL
- Identity: LZ1442.3, Benzethonium chloride
- Chemical name: Benzethonium chloride
- Appearance: White powder
- Storage conditions: Room temperature
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Batch No.: OC0509
- Expiry date: 7 June2002
- Purity: Responsibility of sponsor
- Date received: 20 August 2001
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

At the start of the definitive test, two samples (20 ml) were taken from the freshly-prepared control and test media remaining in the preparation flasks; after 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. On each occasion, one of the samples was analysed immediately and the other was stored in a refrigerator in case further analysis was required.

Vehicle:

no

Details on test solutions:

The test substance (10 mg) was dissolved in culture medium (100 ml) in a volumetric flask. The contents of the flask were shaken vigorously and then serially diluted to provide the test media. An aliquot (3.38 ml) of algal inoculum was added to the test medium (500 ml) at each concentration to give an initial cell density of 1 x 10E04 cells/ml. The required volume (100 ml) of the appropriate inoculated test medium was added to each flask. The definitive test concentrations, selected based on the results of the rangefinding test, were 0.0427, 0.0939, 0.207, 0.455 and 1 mg/l as nominal concentrations.

Test organisms (species):

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

Details on test organisms:

Source:
Axenic, uni-cellular, aqueous slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Cumbria, UK and arrived on 27 July 2001.

Pre-culture:
The aqueous slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium (Appendix 1) was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 ml) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 1.5 x 10E06 cells/mi. Aliquots of this culture were used in the study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

not measured

Test temperature:

The temperature of the incubator ranged between 22.9 and 23. 7°C.

pH:

The pH-value ranged between 6.6 and 10.

Dissolved oxygen:

not measured

Salinity:

freshwater used

Nominal and measured concentrations:

The definitive test concentrations, selected based on the results of the rangefinding test, were: nominal concentrations of 0.0427, 0.0939, 0.207, 0.455 and 1 mg/l. At the start of the test, at 0.207 to 1 mg/l the measured levels of Benzethoniumchloride in samples of the test cultures ranged between 85 and 92% of their nominal values. At the two lowest concentrations, 0.0427 and 0.0939 mg/l, measured levels were close to or below the limit of quantification of the analytical method; estimates of measured concentrations were between 51 and 52 % of their nominal values. After 72 hours, the measured levels were lower, ranging between 42 and 64% of their nominal values. The decreases in the measured levels after 72 hours were thought to be due to the presence of algal cells; losses being greater where cell growth was highest. The overall
geometric mean measured levels of Benzethoniumchloride were 0.0184, 0.0380, 0.124, 0.318 and 0.766 mg/l.

Details on test conditions:

Environmental conditions
Conical flasks (250 ml) each containing 100 ml of control or test culture were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium for 72 hours under continuous illumination of approximately 6650 lux provided by 6 x 30 W "cool white" 1 metre fluorescent tubes. The temperature was maintained at 23 ±2°C.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (95% confidence limits 0.038 and 0.124 mg/l)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

(95% confidence limits 0.23 and 0.38 mg/l)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The results of the most recent laboratory reference test using potassium dichromate indicated that its 72-hour EbC50 to Selenastrum capricomutum was 0.75 mg/l; this was within the range typically obtained in this laboratory (0.3 to 1 mg/l}.

Reported statistics and error estimates:

Dunnett's multicomparison test

Validity criteria fulfilled:

yes

Conclusions:

The EC50-values to Selenastrum Capricornutum were found to be EbC50 (72h): 0.12 mg/l (area under the growth curve (biomass)) and ErC50 (0 -72 h): 0.30 mg/l (average specific growth rate). The corresponding NOEC's were: NOEbC (72h): 0.038 mg/l and NOErC (0 -72h): 0.124 mg/l

Executive summary:

The effect of Benzethoniumchloride on the growth of the unicellular green alga Selenastrum capricornutum was assessed under non-axenic conditions. The study was conducted in accordance with EC Method 3 "Algal Inhibition Test" and the OECD Guideline for Testing of Chemicals No. 201 "Alga, Growth Inhibition Test". Triplicate algal cultures, with an initial cell density of 1 x 104/ml, were exposed to the test item dispersed in algal nutrient medium at nominal concentrations of 0.0427, 0.0939, 0.207, 0.455 and 1 mg/l. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.9 to 23.7°C for 72 hours. At the start of the test, at 0.207 to 1 mg/l the measured levels of Benzethoniumchloride in samples of the test cultures ranged between 85 and 92% of their nominal values. At the two lowest

concentrations, 0.0427 and 0.0939 mg/l, measured levels were close to or below the limit of quantification of the analytical method; estimates of measured concentrations were between 51 and 52 % of their nominal values. After 72 hours, the measured levels were lower, ranging between 42 and 64% of their nominal values. The decreases in the measured levels after 72 hours were thought to be due to the presence of algal cells; losses being greater where cell growth was highest. The overall geometric mean measured levels of Benzethoniumchloride were 0.0184, 0.0380, 0.124, 0.318 and 0.766 mg/l. The test results are expressed in terms of measured concentrations as area under the growth curve (biomass) and growth rate. The following values were derived from the data:

Area under the growth curve (biomass):

- EbC50 (72h): 0.12 mg/l

- NOEbC (72h): 0.038 mg/l

Average specific growth rate:

- ErC50 (0 -72 h): 0.30 mg/l

- NOErC (0 -72h): 0.124 mg/l

Description of key information

The effect of Benzethoniumchloride on the growth of the unicellular green alga Selenastrum capricornutum was assessed under non-axenic conditions. The study was conducted in accordance with EC Method 3 "Algal Inhibition Test" and the OECD Guideline for Testing of Chemicals No. 201 "Alga, Growth Inhibition Test". The EC50-values to Selenastrum Capricornutum were found to be EbC50 (72h): 0.12 mg/l (area under the growth curve (biomass)) and ErC50 (0 -72 h): 0.30 mg/l (average specific growth rate). The corresponding NOEC's were: NOEbC (72h): 0.038 mg/l and NOErC (0 -72h): 0.124 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

0.12 mg/L

EC10 or NOEC for freshwater algae:

0.038 mg/L

Additional information

Source: GLP-report