Chlorhexidine dihydrochloride

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:

2002

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)

Version / remarks:

cited as 92/69/EEC, C.3. (1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Infracor Chemistry Services

Analytical monitoring:

yes

Details on sampling:

The analyses were carried out by measurement of dissolved organic carbon.
The test item concentration range for the testing was below the detection limit of 1 mg TOC/L. Beside this, no suitable substance specific method exist at this time to analyse the test item concentrations in this very low range.
Due to these facts, only the test item concentrations of the stock solution and one of the additional test concentration directly above the detection limit (= additional stability control) could be determined after 0 and 72 h by TOC analysis. These measurements were used to assess the stability of the test item in this test system. The results of the stock solution analysis were used for the calculation of the nominal amount of the test item in the test vials.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus (CHODAT)
- Strain: 86,81 SAG
- Source: Albrecht von Haller-Institut für Pflanzenwissenschaften of the University of Göttingen, Abteilung Experimentelle Phykologie, Nikolausberg 18, Göttingen, Germany
- Age of inoculum (at test initiation): 3 days of preculture prior to the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.2 - 24.7 °C

pH:

At the beginning of the test: 8.4 - 8.9
At the end of the test: 8.0 - 10.0
In some of the test concentrations and in the controls an increase of the pH-value was observed. Since the growth was not influenced by this effect, it was not regarded as important for the quality of the test.

Nominal and measured concentrations:

Initial concentrations of test substance: 0.004, 0.008, 0.016, 0.032, 0.064, 0.12, 0.24 mg/L
The measured recovery rate of test substance concentrations of the stability control (3 mg/L) and of the stock solution (100 mg/L) after 0 hours amounted to more than 80 % of the nominal values. The analytical concentrations of the stock solution and of the stability control after 72 hours did not deviate by more than 20 % from the freshly prepared solutions. It can be therefore con cluded that the test substance concentration was stable during the whole exposure time.
Therefore, the nominal concentration of the stock solution was used to calculate the test substance concentrations in the test vials.

Details on test conditions:

TEST SYSTEM
- Test vessel: The algae were cultured in sterile Erlenmeyer vessels under light benches on a rotary shaker at 100 rpm (12 x 15 minutes/day)
- Type (delete if not applicable): open.Test substance is not volatile
- Control end cells density: Approx. 20,000 cells/mL

GROWTH MEDIUM
- Standard medium used: As described in Guideline 92/69/EEC C.3. (1992) and OECD test Guideline 201 (1984)

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test (0, 72 h)

OTHER TEST CONDITIONS
- Photoperiod: No data (Guideline requires continuous illumination)
- Light intensity and quality: Approx. 6000 - 10000 lux, white

EFFECT PARAMETERS MEASURED:
Cell multiplication inhibition (photometric measurement at 685 nm); calculation of growth rate and biomass production. Cell numbers were determined photometrically after 24, 48 and 72 hours.

Reference substance (positive control):

no

Remarks:

not required by Guideline

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.062 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.016 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Reported statistics and error estimates:

The NOEC-value was determined on the basis of the "area under the growth curve" during the whole exposure time (72 h) using the student t-test.
The percentage growth inhibition rates for each concentration (referred to the control) were calcula ted by probit transformation of the percentage inhibition rates over the regression line. These probits were the basis for the following probit analysis according to Cavalli-Sforza (1972).

Percentage inbibition rates

Concentration [mg/L]	Area under growth curve	Growth rate µ 0-72 h
0.004	-18.5	-4.8
0.008	-18.5	-4.8
0.016	3.7	0.0
0.032	54.9	26.4
0.064	80.2	54.0
0.12	93.8	82.2
0.24	96.1	89.6

 

Validity criteria fulfilled:

yes

Conclusions:

Chlorhexidine dihydrochloride was found to be acutely very toxic towards algae. The 72h-ErC50 and the 72h-ErC10 were determined to be 0.062 mg/L and 0.020 mg/L, respectively.

Description of key information

A GLP growth inibition study with Desmodesmuss subspicatus according to the OECD 201 guideline was performed. Chlorhexidine dihydrochloride was found to be acutely very toxic towards algae. The 72h-ErC50 and the 72h-ErC10 were determined to be 0.062 mg/L and 0.020 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.062 mg/L

EC10 or NOEC for freshwater algae:

0.02 mg/L