Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

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:

14-May-2012 to 16-May-2012

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision: 19-November-2010

Analytical monitoring:

no

Details on test solutions:

Based on the Sponsor’s information, no stock solution could be prepared due to the low water solubility of the test item.

Therefore, test item amounts of 5.04, 50.11, 500.2, 500.1 and 500.1 mg were weighed by means of an analytical balance onto glass slides and transferred into the designated test vessels. Then, 234 mL water was added to each vessel.

The test item was mixed into the water by intense stirring for 3 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.

After stirring, the appearance of the test media was evaluated, and all test concentrations were clearly above the water solubility limit of C.I. Leuco Sulphur Brown 96 under the test conditions. The pH of the suspensions was adjusted to the range 7.6-7.9 with a diluted sodium hydroxide solution. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.

Test organisms (species):

activated sludge

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 6.9 to 7.5 with a diluted sodium hydroxide solution.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

not applicable.

Hardness:

not applicable.

Test temperature:

The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period.

pH:

For Information on the pH please see attached Table 1.

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 60-70% saturation and the sludge flocs were maintained in suspension during the incubation period. Just before measurement of the respiration rates the dissolved oxygen concentrations were at least 7.8 mg/L .

Salinity:

according to guideline.

Nominal and measured concentrations:

A range-finding test using the following three test item concentrations 10 mg/L, 100 mg/L and 1000 mg/L was performed. Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration three replicates were prepared.

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).

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

Details on test conditions:

During the incubation period of 3 hours all vessels were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item. The concentration of dissolved oxygen did not drop below 60-70% saturation and the sludge flocs were maintained in suspension during the incubation period. Just before measurement of the respiration rates the dissolved oxygen concentrations were at least 7.8 mg/L (see attached Table 1). The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period.

Distilled or deionized water, containing less than 1 mg/L dissolved organic carbon was used except where chlorine free tap water is specified.

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

The test was performed in 2000 mL glass beakers containing 500 mL of incubation mixture. The test vessels were labeled with the necessary information to ensure unique identification.

At the start of the test, 16 mL synthetic sewage (see Section 3.4) were made up to 250 mL with water and 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to this first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage was added to the appropriate amounts of reference item or test item in water to reach a volume of 250 mL, followed by the addition of 250 mL inoculum.

The addition of the inoculum to the incubation mixture is considered as the start of the 3-hour incubation time period.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

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

Remarks:

respiration rate

Details on results:

Toxicity of C.I. Leuco Sulphur Brown 96
The influence of C.I. Leuco Sulphur Brown 96 on the respiration rate of activated sludge is presented in the attached Table 1 and Figure 1.

Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of C.I. Leuco Sulphur Brown 96 under the test conditions) was reached at a concentration of 10 mg/L.

At all tested concentrations up to and including 1000mg/L, the inhibition of the respiration rate of activated sludge after the incubation period of three hours was in the range 0.6 to 6.4% compared to the control. For the three replicates of the highest test concentration of 1000 mg/L the mean inhibition of the respiration rate was calculated to be 3.6%, compared to the control. According to the results of a Welch t-test (one sided smaller, α = 0.05), the test item C.I. Leuco Sulphur Brown 96 had no statistically significant inhibitory effect on the respiration rate of up to and including the highest test item concentration of 1000 mg/L.

Thus, the 3 hour NOEC of C.I. Leuco Sulphur Brown 96 to activated sludge micro-organisms was at the saturation concentration of the test item under the test conditions, or at 1000 mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3 hour EC10, EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

In conformity with the guideline, since in the range-finding test the test item showed at the highest tested concentration (performed as triplicate) no inhibition a definitive test is unnecessary.


Toxicity of 3,5-Dichlorophenol (Positive Control)
The results are presented in the attached Table 1 and Figure 1.

The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 7.7 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3 hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming suitability of the activated sludge used.

Respiration Rates of the Blank Controls
The results are presented in the attached Table 1.

The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 47 and 50 mg O2 per dry weight of activated sludge per hour and for the two blank controls prepared at the end of the test were 47 and 46 mg O2/gh. All four values are higher than the 20 mg O2/gh stipulated in the guideline.

The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 3% and is therefore, as stipulated in the guideline, not more than 30%.

Results with reference substance (positive control):

The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 7.7 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3 hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The NOEC was derived by per-vessel statistics using the Welch t-test. Compared were the three replicates of the highest test item concentration with the 4 blank control replicates using one-tailed (smaller) hypothesis and α = 0.05.

The 3 hour EC10, EC20, EC50 and EC80 values of the test item C.I. Leuco Sulphur Brown 96 and their 95% confidence limits could not be calculated because of the absence of a toxic effect.

The 3 hour EC50 of the reference item 3,5 dichlorophenol was calculated by Probit Analysis.

All test results were based on nominal concentrations of the test and reference item.

Validity of the Test:

The test is regarded as valid since:

 

- the specific respiration rate of the blank controls was in the range 46 to 50 mg oxygen per gram dry weight of sludge per hour and therefore, higher than 20 mg O₂/gh.

 

- the coefficient of variation of oxygen uptake rate in control replicates was 3% and therefore, less than 30%.

 

- the EC₅₀(3-Hour) of the reference item 3,5-dichlorophenol was 7.7 mg/L and therefore, in the range 2 to 25 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The test item C.I. Leuco Sulphur Brown 96 has no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to and including the test item concentration of 1000 mg/L.

Executive summary:

The inhibitory effect of the test item C.I. Leuco Sulphur Brown 96 on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3‑hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010.

 

A range-finding test using the following three test item concentrations 10 mg/L, 100 mg/L and 1000 mg/L was performed. Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration three replicates were prepared.

 

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).

 

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

 

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

 

The results of the blank and positive control treatments confirmed suitability of the activated sludge and the method used. The validity criteria were fulfilled.

 

The test item C.I. Leuco Sulphur Brown 96 had no statistically significant inhibitory effect on the respiration rate of activated sludge at all tested concentrations up to and including 1000 mg/L after the incubation period of three hours.

 

Thus, the 3‑hour NOEC of C.I. Leuco Sulphur Brown 96 to activated sludge micro-organisms was 1000 mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3‑hour EC₁₀, EC₂₀, EC₅₀and EC₈₀could not be calculated, but were clearly higher than 1000 mg/L.

 

In conformity with the guideline, since in the range-finding test the test item showed at the highest tested concentration (performed as triplicate) no inhibition of the respiration rate a definitive test is unnecessary.

Description of key information

The test item C.I. Leuco Sulphur Brown 96 has no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to and including the test item concentration of 1000 mg/L. Thus, the 3-hour NOEC of C.I. Leuco Sulphur Brown 96 to activated sludge micro-organisms was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The inhibitory effect of the test item C.I. Leuco Sulphur Brown 96 on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3‑hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010.

A range-finding test using the following three test item concentrations 10 mg/L, 100 mg/L and 1000 mg/L was performed. Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration three replicates were prepared.

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). Four blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item were included in the range-finding test. The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), the test item group (with ascending concentrations) and at last again two blank control vessels.

The results of the blank and positive control treatments confirmed suitability of the activated sludge and the method used. The validity criteria were fulfilled.

The test item C.I. Leuco Sulphur Brown 96 had no statistically significant inhibitory effect on the respiration rate of activated sludge at all tested concentrations up to and including 1000 mg/L after the incubation period of three hours. Thus, the 3-hour NOEC of C.I. Leuco Sulphur Brown 96 to activated sludge micro-organisms was 1000 mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3‑hour EC10, EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

In conformity with the guideline, since in the range-finding test the test item showed at the highest tested concentration (performed as triplicate) no inhibition of the respiration rate a definitive test is unnecessary.