4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

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:

June 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test concentration was verified by chemical analysis. Water samples (100 rnl in duplicate) were taken from solvent control and test cultures at 0 hours and at 72 hours (replicates pooled) and sent to the analytical laboratory of the test institute. At 0 hours, additional samples were also taken from the 10 mgll dispersion in algal medium and either filtered or centrifuged prior to analysis. The filtered samples were passed through two in-line filters (0.22 um) to remove any particulate matter prior to analysis. The centrifuged samples were spun down at a speed of 4000 n/min Exp.-1 for 30 minutes prior to analysis. Additional flasks containing cultures of the 10 mg/l exposure level were incubated with the test vessels in order to provide sufficent sampling volume at 72 hours. At 72 hours, additional samples were taken from test and satellite vessels (replicates pooled), in order to obtain information on the extent of adsorption / absorption of the test substance by the algal cells and either filtered or centrifuged prior to analysis, as outlined above.

Vehicle:

yes

Details on test solutions:

The test substance was dissolved in an auxiliary solvent, 10% Tween 80 dimethylformamide, to give a preliminary stock solution of 100 mglml. An aliquot (200 ul) of this stock solution was added to 2 litres of algal medium to give the final test concentration of 10 mg/l. A subsample (200 ml) of the 10 mg/l dispersion was incubated under the conditions of the test in order to provide a comparison between cultures with and without algal cells present. The exposure level was achieved by adding 4 ml of concentrated algal suspension to one litre of the 10 mg/l test dispersion. The control was prepared by adding 4 ml of the concentrated algal inoculum to one litre of sterile nutrient medium. The solvent control was prepared as for the control but with an aliquot (100 ul) of auxiliary solvent added to one litre of the control preparation.

Test organisms (species):

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

Details on test organisms:

Algae were supplied by Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, UK. Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination (2: 7000 lux) and aeration via narrow bore glass tube at 24°C to give an algal
suspension in log phase growth characterised by a cell density of 2.4 x 10 Exp.6 cells per ml. The suspension was concentrated by centrifugation to give a cell density of 9.7 x 10 Exp.6 cells per ml prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not calculated

Test temperature:

24°C

pH:

7.7 - 10.0

Dissolved oxygen:

Not available

Salinity:

Freshwater used

Nominal and measured concentrations:

- Nominal test concentration: 10 mg/l
- Mean measured concentration: 7.4 mg/l
One test concentration was used and one solvent control (100 ul auxiliary solvent per litre) of six replicates each, and one untreated control in triplicate.

Details on test conditions:

Conical flasks (250 ml) each containing 100 ml of test or control culture were loosely capped with foil and placed at random in a Gallenkarnp Illuminated Orbital Incubator. The cultures were incubated, without media renewal, for 72 hours under continuous illumination of approximately 7000 lux provided by 7 x 30 W "universal white" 1 metre fluorescent tubes. The temperature was maintained at 24°C and gaseous exchange and suspension of the algal cells was ensured by the action of the orbital shaker oscillating at 120 cycles per minute.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL could not be calculated

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL could not be calculated

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL could not be calculated

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL could not be calculated

Results with reference substance (positive control):

No positive control tested

Reported statistics and error estimates:

Reference: Bartlett, M.S., 1937, Properties of sufficiency and statistical tests. Proceedings of the Royal Society. Series A, 160, 268 - 282).

The analytical results are expressed in terms of the mean measured concentration, which was 74% of nominal in this study. The measured concentration of the test exposure level was 88% of nominal at the start of the study. Over the course of the study, approximately 25% was lost from dispersion, with 63% of nominal remaining at 72 hours. This loss of test substance from dispersion was also reflected in the measured concentrations of the test dispersion, incubated without algal cells present (32% of nominal lost over 72 hours cf: 25%). This indicates that the presence of algal cells did not affect the stability of the test dispersion. The losses of test substance from dispersion over the duration of the study are considered to be due to the inherent difficulties in maintaining a test dispersion at a concentration well in excess of the water solubility of the test substance.

The maximum concentration employed in this study was nominally 10 mg/l, the highest concentration at which it was possible to obtain a visually stable test dispersion. It was considered unnecessary and unrealistic to test at concentrations in excess of this value given the low solubility of the test substance in water and having regard for the maximum amount of auxiliary solvent permitted under the constraints of this study. The apparent instability indicated by comparison of the fresh and expired solutions for the filtered and centrifuged samples would suggest that the presence of algal cells has compromised the effectiveness of these methods for determination of the amount of P5367 truly in solution.

Validity criteria fulfilled:

yes

Conclusions:

The EbC50 and ErC50 were determined to be > 1 mg/l (measured concentration of filtered algal media). If the results are expressed as mean measured concentration of the test dispersion, the ErC50 and EbC50 were both > 7.4 mg/l.

Executive summary:

The study was performed in 1996 as GLP-test following EU-testing method C.3 on the green algae species Selenastrum capricornutum. The test was performed under static conditions as limit test with a nominal test item concentrations of 10.0 mg/l in the main study. Due to the low solubility, the test item was dissolved in an auxiliary solvent, 10% Tween 80 dimethylformamide mixture to give a preliminary stock solution of 100 mg/ml. The loss of concentration of test item over the test period was 26%, therefore 74% of the nominal concentration was found at the end of the study.

Statistically significant inhibition of growth (21 %) was found in the test cultures, when compared to the biomass of the solvent control using the student's t-test. Bartlett's test for homogeneity confirms the difference was not due to replicate variation. However, since the percentage inhibition was less than 25% and the concentration of the test dispersion is well in excess of the water solubility of the test substance (2.5 x 10 Exp.-5 g/l) this is not considered to be biologically significant.

Description of key information

The study was performed in 1996 as GLP-test following EU-testing method C.3 on the green algae species Selenastrum capricornutum. The test was performed under static conditions as limit test with a nominal test item concentrations of 10.0 mg/L in the main study. Due to the low solubility, the test item was dissolved in an auxiliary solvent, 10% Tween 80 dimethylformamide mixture to give a preliminary stock solution of 100 mg/ml. The loss of concentration of test item over the test period was 26%, therefore 74% of the nominal concentration was found at the end of the study. Statistically significant inhibition of growth (21 %) was found in the test cultures, when compared to the biomass of the solvent control using the student's t-test. Bartlett's test for homogeneity confirms the difference was not due to replicate variation. However, since the percentage inhibition was less than 25% and the concentration of the test dispersion is well in excess of the water solubility of the test substance (2.5 x 10 Exp.-5 g/L), this is not considered to be biologically significant.

The EbC50 and ErC50 were determined to be > 1 mg/L (measured concentration of filtered algal media). If the results are expressed as mean measured concentration of the test dispersion, the ErC50 and EbC50 were both > 7.4 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.4 mg/L

Additional information

Source: GLP-report