4-chloro-3,5-xylenol

Toxicity to aquatic algae and cyanobacteria

Administrative data

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:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-3,5-xylenol

EC Number:

201-793-8

EC Name:

4-chloro-3,5-xylenol

Cas Number:

88-04-0

Molecular formula:

C8H9ClO

IUPAC Name:

4-chloro-3,5-dimethylphenol

Test material form:

solid: crystalline

Details on test material:

crystaline

Specific details on test material used for the study:

PCMX
cream coloured crystalline solid
285/13847
27 September 2001
room temperature, in the dark

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of
Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa
(CCAP), Institute of Freshwater Ecology.

Test solutions

Vehicle:

not specified

Details on test solutions:

An amount of test material (100 mg) was dissolved m culture medium with the aid of
ultrasonication (approximately 10 minutes) followed by high sheer mixing (7500 rpm for
approximately 10 minutes) and the volume adjusted to 1 litre to give a 100 mg/l stock solution. A
series of dilutions were made from this stock solution to give further stock solutions of 20, 10,
5.0, 2.5 and 1.25 mg/l. An aliquot (500 ml) of each of the 1.25, 2.5, 5.0, 10 and 20 mg/l stock
solutions was separately mixed with algal suspension (500 ml) to give the required test
concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/l.

Test organisms

Test organisms (species):

Scenedesmus sp.

Details on test organisms:

Scenedesmus subspicatus is a freshwater unicellular alga, representative of primary producers
found in natural waters and can therefore be considered as an important non-target organism in
freshwater ecosystems.

Study design

Test type:

static

Water media type:

not specified

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

. After 72 hours there were no abnormalities detected in the control or test cultures at 0.625, 1.25 and 2.5 mg/l, however few intact cells were observed to be present in the test cultures at 5.0 and 10 mg/l.

Post exposure observation period:

At the start of the test the control and all test cultures were observed to be clear colourless
solutions. After the 72-Hour test period the control, 0.625, 1.25 and 2.5 mg/l test cultures were
observed to be bright green dispersions whilst the 5.0 and 10 mg/l test cultures were observed to
be clear colourless solutions.

Test conditions

Test temperature:

Temperature was maintained at
24 ± 1 °C throughout the test.

pH:

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.4 at
0 hours to pH 7 .6 - 7. 7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH
units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Analysis of the test solutions at 0 and 72 hours (see Appendix 2) showed measured test
concentrations to range from 98% to 110% of nominal values and so it was considered justifiable
to calculate the EC50 values in terms of the nominal test concentrations only.

Details on test conditions:

Exposure conditions
As in the range-finding test 250 ml glass conical flasks were used. Three flasks each containing
100 ml of solution were prepared for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell
density of 3 .66 x 106 cells per ml. This suspension was diluted to a cell density of 2.16 x 104 cells
per ml prior to use. At initiation of the test the culture contained a nominal cell density of 104 cells
per ml.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron®
Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000
lux) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter®
Multisizer II Particle Counter.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.8 - ca. 3.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: . The No Observed Effect Concentration at 72 hours was 2.5 mg/l.

Details on results:

Analysis of the test solutions at 0 and 72 hours (see Appendix 2) showed measured test
concentrations to range from 98% to 110% of nominal values and so it was considered justifiable
to calculate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated
over a 72-Hour period and gave an EbCso (72 h) value of 3.8 mg/l and an ErCso (0 - 72 h) value
of 3.6 mg/l. The No Observed Effect Concentration at 72 hours was 2.5 mg/l.

Any other information on results incl. tables

Nominal Concentration

pH Cell Densities* (cells per ml) pH

(mg/I) Oh Oh 24h 48 h 72h 72h

Control R1 7.4 l.28E+04 2.18E+04 l.12E+05 6.43E+05 7.6

Rz 7.4 l.29E+04 2.92E+04 6.58E+04 5.66E+05 7.7

R3 7.4 l.28E+04 2.31E+04 6.63E+04 5.10E+05 7.7

Mean l.28E+04 2.47E+04 8.15E+04 5.73E+05

0.625 R1 7.3 l.25E+04 2.62E+04 7.72E+04 5.93E+05 8.0

Rz 7.3 l.21E+04 2.12E+04 8.45E+04 5.02E+05 8.0

R3 7.3 l.29E+04 2.78E+04 l.01E+05 6.19E+05 8.0

Mean l.25E+04 2.51E+04 8.75E+04 5.71E+05

1.25 Ri 7.3 l.24E+04 3.40E+04 l.11E+05 7.58E+05 8.6

Rz 7.3 l.14E+04 2.64E+04 8.70E+04 7.05E+05 8.4

R3 7.3 l.29E+04 3.03E+04 l.18E+05 6.04E+05 8.4

Mean l.22E+04 3.02E+04 l.05E+05 6.89E+05

2.5 Ri 7.3 l.24E+04 3.41E+04 l.05E+05 7.10E+05 7.9

Rz 7.3 l.22E+04 3.16E+04 l.02E+05 6.91E+05 8.0

R3 7.3 l.29E+04 3.12E+04 l.09E+05 8.69E+05 8.4

Mean l.25E+04 3.23E+04 l.05E+05 7.56E+05

5.0 Ri 7.2 l.28E+04 8.84E+03 l.64E+04 l.63E+04 7.4

Rz 7.2 l.20E+04 7.60E+03 l.49E+04 9.46E+03 7.4

R3 7.2 l.25E+04 7.51E+03 l.51E+04 l.05E+04 7.4

Mean l.24E+04 7.98E+03 l.54E+04 l.21E+04

10 R1 7.2 l.26E+04 6.10E+03 l.07E+04 4.09E+03 7.3

Rz 7.2 l.25E+04 6.14E+03 l.12E+04 4.28E+03 7.3

R3 7.2 l.09E+04 7.03E+03 l.59E+04 4.42E+03 7.3

Mean l.20E+04 6.42E+03 l.26E+04 4.26E+03

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated
over a 72-Hour period and gave an EbCso (72 h) value of 3.8 mg/l and an ErCso (0 - 72 h) value
of 3.6 mg/l. The No Observed Effect Concentration at 72 hours was 2.5 mg/l.

Executive summary:

From the data given in Tables 2 and 3, it is clear that both the growth (r) and the biomass (b) of

Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over

the 72-Hour exposure period.

The percentage inhibition values, (IA) values (Table 3) were plotted against test concentration

(Figure 2), a line fitted by eye and the EC50 value with respect to the area under the growth curve,

EbCso (72 h) read from the graph.

Percentage reductions in growth rate and the EC50 value with respect to growth rate, ErCso value

(0 - 72 hours) were calculated as in Section 3.3.3.1 and the results given in Table 3 and Figure 2.

Accordingly the following results were determined from the data:

EbCso (72 h)

ErCso (0 - 72 h)

: 3.8 mg/I*

: 3.6 mg/I*

* It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the

models available for the calculation of confidence limits eg Litchfield and Wilcoxon, Probit, Logistic and

Weilbulls models (Litchfield and Wilcoxon 1949).

SPL PROJECT NUMBER: 905/042 PAGE 13

where EbCso is the test concentration that reduced biomass by 50% and ErCso is the test

concentration that reduced specific growth rate by 50%.

Statistical analysis of the area under the growth curve data was carried out for the control and all

test concentrations using one way analysis of variance incorporating Bartlett's test for

homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure

for comparing several treatments with a control (Dunnett 1955). There were no statistically

significant differences between the control, 0.625, 1.25 and 2.5 mg/l test concentrations (P;:::0.05),

however all other test concentrations were significantly different (P<0.05) and, therefore the "No

Observed Effect Concentration" (NOEC) was 2.5 mg/l.

The following data show that the cell concentration of the control cultures increased by a factor of

45 during the test in line with the OECD Guideline that states the enhancement must be at least by

a factor of 16 after 72 hours:

Mean cell density of control at 0 hours

Mean cell density of control at 72hours

5.2.2 Observations on cultures

1.28 x 104 cells per ml

5. 73 x 105 cells per ml