N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2014 to 20 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Remarks:

The test item was mixed into the tap water by intense stirring for 1 hour at room temperature to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.

The sludge was washed twice with chlorine-free tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and readjusted to the level of 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge inoculum was adjusted from 6.8 to 7.7 with a 1M NaOH solution.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

not specified

Test temperature:

20°C (measured in one control at start and end)

pH:

Test start: 7.4 to 7.5
Test end: 7.8 to 8.0

Dissolved oxygen:

Test start: 7.5 to 8.2 mg O2/L
Test end:6.4 to 7.3 mg O2 /L

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

The nominal test concentrations were 10.0, 31.6, 100, 316 and 1000 mg/L

Details on test conditions:

Test vessels: 2000 mL glass beakers containing 500 mL of incubation mixture
Replication: Three replicates per test concentration
Aeration: Continuous aeration with compressed air
Lighting: Continuous illumination

Reference substance (positive control):

yes

Remarks:

3, 5-dichlorophenol at 2, 10 and 32 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. The results of the controls (difference of the oxygen consumption rates of the two controls: 5%) and the reference item 3 hour EC50 value demonstrated the suitability of the sludge used.
The 3-hour EC10, EC20, EC50 and EC80 values and their 95% confidence limits were derived by Probit Analysis using linear maximum likelihood regression. The 3 hour EC50 of the reference item 3,5 dichlorophenol was calculated by Weibull Analysis using linear maximum likelihood regression.


Nominal concentration Oxygen consumption rate Inhibition (%)
(mg ai/L) (mg O2 /Lh)


control 64.41 -
10 64.28 0.2
31.6 60.58 6.2
100 53.42 17.0
316 47.24 26.7
1000 39.92 38.0

- % Inhibition = increased oxygen consumption rate relative to that of control

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 11 mg/L (the 95% confidence limits could not be calculated for mathematical reasons).
The 3-hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming suitability of the activated sludge used.

Validity criteria fulfilled:

yes

Conclusions:

A clear concentration-effect relationship on the inhibition of the respiration rate was determined. The 3 hour EC10, EC20, EC50, and EC80 were determined to be 51, 185, >1000 and > 1000 mg /L respectively. The NOEC was determined to be 31.6 mg /L.

Executive summary:

The inhibitory effect of the test item CA5204A on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a three-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010.

In conformity with the guideline a definitive test was performed, since in the range-finding test the test item showed statistically significant inhibition of the respiration rate.

Based on the results of the  range-finding test a definitive test with the following nominal test item concentrations was performed: 10.0, 31.6, 100, 316 and 1000 mg/L, with three replicates per concentration.

As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).

Six blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the definitive test.

The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and finally two blank control vessels.

The respiration rate of the activated sludge at the lowest tested concentration of 10 mg/L was similar to the control (0.2%). At the next higher test concentration of 31.6 mg/L the respiration was inhibited by 6.2% compared to the control. Although statistically significant, this low inhibition is considered as biologically not relevant (i.e. less than 10%). At the higher test item concentrations of 100, 316 and 1000 mg/L the inhibitory effect increased and reached 17%, 27% and 38%, respectively. A clear concentration-effect relationship on the inhibition of the respiration rate was determined.

The 3-hour NOEC and ECX-values and their 95% confidence limits were as follows:

NOEC 31.6 mg/L

EC10 51 mg/L 95% confidence limits: 14 – 97 mg/L

EC20: 185 mg/L 95% confidence limits: 96 – 287 mg/L

EC50: >1000 mg/L

EC80: >1000 mg/L

Description of key information

A clear concentration-effect relationship on the inhibition of the respiration rate was determined. The 3 hour EC₁₀, EC₂₀, EC₅₀, and EC₈₀were determined to be 51, 185, >1000 and > 1000 mg /L respectively. The NOEC was determined to be 31.6 mg /L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

31.6 mg/L