3,5-xylidine

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:

26 Jan 1998 - 31 March 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study on behalf of competent authorities of Japan,

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

200 mg of the test substance was weighed, dissolved in medium, and made up to 100 mL volume to prepare a stock solution with a concentration of 2000 mg/L. Only culture medium was used for the control treatments.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection, June 20, 1996
- cultured in lab

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

63 mg/L (CaCO3 equivalent)

Test temperature:

23±2 ℃

pH:

The pH of the test solution ranged from 7.8 to 7.9 at the beginning of the exposure and from 8.0 to 10.5 at the end of the test.

Nominal and measured concentrations:

control, 2.00, 3.40, 5.80, 10.0, 17.0, 29.0, 50. 0 mg/L (ratio: 1.7)

Details on test conditions:

TEST SYSTEM
- Test vessel: triangular flask with stopper; AGP-150RL model
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 100mL
- Aeration: shaking culture (100 rpm)
- Initial cells density: 1x10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes, OECD medium

- Culture medium different from test medium: no
- Intervals of water quality measurement: 3 times a week

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 4000 - 5000 lux (within ±20% variation, near the flask surface)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell concentration was measured at 24, 48 and 72 hours with particle counter (CDA-500) after mixing 1 mL test solution with 9 mL electrolyte solution. The pH at the time of preparation of the test solution was measured for one preliminary bottle prepared separately from the triplicate, and the pH at the beginning of exposure in each concentration zone was used as the pH at the end of exposure.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study
- Test concentrations: 2.0, 5.0, 10.0, 20, 50 ,100, 200 mg/L
- Results used to determine the conditions for the definitive study: Growth rate 92% and 100% at 5 and 5 mg/L, 55% at 20 mg/l and 4% at 50 mg/L

Reference substance (positive control):

yes

Remarks:

The growth inhibition concentration (EbC50) of the reference material (potassium dichromate, special grade) for 72 hours was 0.410 mg/L (linear regression analysis).

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.3 mg/L

95% CI:

>= 19.8 - <= 25.1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

area under growth curve

Details on results:

Since the concentration of each test solution was within ±20% of the set value at the start of the test, the set value was used to calculate the following growth inhibition concentrations.
The percentage of the set value after 72 hours of exposure was 92% to 95%.

Growth curve
　Cell concentrations in the control increased an average of 50-fold after 72 hours of incubation, indicating normal growth under the test conditions.
However, when cultured in a permeable silicone stopper, growth is usually observed more than 100-fold. However, the growth of the cells in the control area increased more than 100-fold under the test conditions, whereas the growth of the cells in the control area was normally observed when the cells were cultured in a ventilated silicon plug.
However, the growth rate was lower than that of the ventilated system, because the growth rate of the volatile substance was lower than that of the ventilated system because the culture was conducted under sealed conditions.

Reported statistics and error estimates:

The 50% growth inhibition concentration (EC50) was calculated using the growth inhibition rates from the area and rate methods calculated. The EC50 value (and its 95% confidence interval whenever possible) was calculated from the intersection of the 50% inhibition rate and a linear regression analysis (least squares method) using the points where linearity was observed. he EbC50 (0-72) was calculated by the area method, and the ErC50 (24-72) was calculated by the rate method.
Bartlett's test of equal variance (α=0.01) was performed to confirm equality of variance, followed by one-way ANOVA (1-way ANOVA, α=0.05) and Williams' multiple comparison test (α=0.05, two-tailed). NOECb (0-72) was determined by the area method, and NOECr (24-48) or NOECr (24-72) where determined by the rate method.

Validity criteria fulfilled:

yes

Conclusions:

The 50% growth inhibitory concentration (EC50) and the maximum no observed effect concentration (NOECr) obtained according to the growth rate during the 72-hour exposure were 33.2 mg/L and 17 mg/L, respectively.

Executive summary:

The GLP growth inhibition test with green algae (Raphidocelis subcapitata, former Selenastrum capricornutum) was conducted for 72 hours under static conditions to determine the 50% growth inhibition concentration (EC50) and the no effect concentration (NOEC) of 3,5-Xylidine according to OECD Guideline 201. The concentrations of the test substance in the test solutions was set to (control), 2.00, 3.40, 5.80, 10.0, 17.0, 29.0, 50. 0 mg/L. The 50% growth inhibitory concentration (EC50) and the maximum no observed effect concentration (NOECr) obtained according to the growth rate during the 72-hour exposure were 33.2 mg/L and 17 mg/L, respectively.

Description of key information

OECD 201, GLP, Raphidocelis subcapitata (former Selenastrum capricornutum), static, 72h: ErC50) 33.2 mg/L, (NOECr) 17 mg/L (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

33.2 mg/L

EC10 or NOEC for freshwater algae:

17 mg/L