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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-May-2012 to 07-Jun-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision: 19-November-2010

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The study was performed with aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic sewage. The sludge was washed twice with 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, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

167 mg/L

Based on:

test mat.

Initial conc.:

15 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of Test Flasks:
One day before test start (Day -1), between 2400 and 2910 mL of untreated test medium was filled into 5 liter flasks (amber glass). To each flask 90 mL activated sludge inoculum was added.

The test media were aerated overnight with CO2-free air to purge the system of carbon dioxide.

On the following day (Day 0), defined amounts of the test item were directly added to the test flasks. No emulsifiers or solvents were used.

The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with CO2-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.

The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL 0.05 M NaOH and the second one containing 200 mL 0.05 M NaOH, were connected in series to the exit air line of each test flask.

Test Conditions:
Test vessels: 5-liter all-glass amber bottles.

Identification: The test vessels were labeled with the necessary information to ensure unmistakable identification.

Test duration: 28 days

Light conditions: The test vessels were incubated in a dark room.

Test temperature: 22-24 °C. The inoculated flasks were incubated in a temperature-controlled room. The temperature was checked on each sampling date in a separate flask with purified water. Additionally, the room temperature was continuously recorded.

CO2-free air: Air was led through a bottle containing about 750 mL of a 2 M NaOH solution to trap CO2. The CO2-free air was passed through the test solutions at a rate corresponding to about 30–100 mL/min.

pH: Prior to test start (Day 0), the pH was measured in each test flask after the addition of test and/or reference item and was found to be 7.4 in all flasks. At the end of incubation (Exposure Day 28), the pH was measured again in each test flask.

For information on test water, test concentrations and Sampling please see section "any other information on materials and methods" below.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

-12.6

Sampling time:

28 d

Details on results:

The measured concentration of inorganic carbon (IC) and the absolute amount of IC produced are presented in the attached Table 1 and Table 2.


Biodegradation of the Test Item
The results are presented in the attached Table 3 and in Figure 1.

The calculation of the percent biodegradation of the test item was based on the carbon content of 0.09 mg C/mg test item provided by the Sponsor.

The CO2 formation of the test item C.I. Leuco Sulphur Brown 96 in the test media was not higher than the CO2 formation of the inoculum controls.

Consequently, C.I. Leuco Sulphur Brown 96 was not biodegradable under the test conditions within 28 days.


Biodegradation of the Reference Item
The results are presented in the attached Table 3 and in Figure 1.

The calculation of the percent biodegradation of the reference item was calculated based on a calculated total organic carbon content (TOC) of 0.58 mg C/mg sodium benzoate.

In the procedure controls, average biodegradation of the reference item was 82% by Exposure Day 14, thus confirming suitability of the activated sludge (≥ 60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 94%.


Biodegradation in the Toxicity Control
The results are presented in the attached Table 3 and in Figure 1.

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total organic carbon content (TOC) of the test item and the reference item.

CO2 formation in the toxicity control showed a slightly delayed course over the 28 day exposure period compared to the two procedure controls, containing only the reference item. Rapid degradation of 30% of the TOC occurred within the first seven days of incubation. Afterwards, biodegradation only increased slightly. Within 14 days of exposure, biodegradation was 32%. By the end of the test (Exposure Day 28), the CO2 formation corresponded to 38% of the TOC.
However, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 167 mg/L because biodegradation in the toxicity control was≥25% within 14 days of incubation.


IC/TC Ratio
The test item was not soluble in test water at the tested concentration of 167 mg/L; therefore, in order to calculate the IC/TC ratio, the nominal TC contribution of the test item, i.e. 15 mg C/L, was added to the TC concentration measured in the test item vessel before the test item addition.

The IC content in the test item vessels was less than 5% of the TC content, therefore fulfilling this validity criterion.


Driving Off CO2
No significant difference was found between the absolute amounts of IC measured on Exposure Day 28 and the absolute amounts of IC measured after acidification on Day 29 (see attached Table 2). Consequently, no significant amount of residual CO2 was present in the test solutions or suspensions at the end of the test.


pH Measurement
The results are presented in the attached Table 4.

The pH measured in all flasks at the end of exposure (Day 28) was between 7.3 and 7.5.

Results with reference substance:

The results are presented in the attached Table 3 and in Figure 1.

The calculation of the percent biodegradation of the reference item was calculated based on a calculated total organic carbon content (TOC) of 0.58 mg C/mg sodium benzoate.

In the procedure controls, average biodegradation of the reference item was 82% by Exposure Day 14, thus confirming suitability of the activated sludge (≥ 60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 94%.

The results are considered valid since the following criteria are met:

 

-      ₂evolution in the inoculum controls at the end of the test was 28 and 33 mg/L (corresponding to 23 and 27 mg C per flask; criterion: 40 mg CO₂/L, corresponding to 33 mg C per flask).

-       The IC content in the test item flasks was 0% of the TC (criterion: less than 5% of the TC).

-       The criterion regarding the difference of duplicate values for the degradation of the test item is not applicable, since no degradation of the test item was observed (criterion less than 20% difference at the plateau, at the end of the test or at the end of the 10‑day window).

-       The percentage degradation of the reference item reached the level for ready biodegradability (>60% in a 10-day window) by Exposure Day 7 (criterion: at least 60% in a 10-day window by Day 14).

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item C.I. Leuco Sulphur Brown 96 was not biodegradable under the conditions of the performed 28 Day CO2 Evolution (Modified Sturm) Test according to the Method C.4-C of Commission Regulation (EC) No 440/2008, and the OECD Guideline for Testing of Chemicals, No. 301 B (1992) and the US EPA Draft Ecological Effects Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Executive summary:

The test item C.I. Leuco Sulphur Brown 96 was investigated for its ready biodegradability in a 28‑Day CO₂Evolution (Modified Sturm) Test according to the Method C.4-C of Commission Regulation (EC) No 440/2008, and theOECD Guideline for Testing of Chemicals, No. 301 B (1992) and the US EPA Draft Ecological Effects Test Guidelines OPPTS 835.3110 (Paragraph (m)).

 

The test item C.I. Leuco Sulphur Brown 96 was not biodegradable under the test conditions within 28 days.

 

In the toxicity control, containing both C.I. Leuco Sulphur Brown 96 and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, C.I. Leuco Sulphur Brown 96 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 167 mg/L.

 

In the procedure controls, average biodegradation of the reference item was 82% by Exposure Day 14, thus confirming suitability of the activated sludge (≥60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 94%.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 July 2012 to 09 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

The following modifications were made:
- Activated sludge from only one source was used.
- The activated sludge was not fed during the holding period.
- The holding period was maximum seven days.
- The test water according to OECD Guideline for Testing of Chemicals No. 301 F, Ready Biodegradability: Manometric Respirometry Test, 1992 was used.
- The test was run at 22 °C.
- Only the biological oxygen demand (BOD) was monitored, no test item specific analysis was performed.

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision: 19-November-2010

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

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 three times with 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, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 100 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

223 - 224 mg/L

Based on:

test mat.

Initial conc.:

51 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test duration: 28 days

Light conditions: Darkness

Test temperature: 22 °C, maintained with a built-in thermostat and checked once per week.

pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculums and again at the end of incubation.

For further information on test water and test concentrations please see section "Any other information on materials and methods" below.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Biodegradation of the test item:
The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 0.23 mg O2/mg test item.

The oxygen consumption of the test item C.I. Leuco Sulphur Brown 96 in the test media was slightly lower than the normal range of the oxygen consumption in the inoculum controls.

Consequently, C.I. Leuco Sulphur Brown 96 was not biodegradable under the test conditions within 28 days.

Biodegredation of the reference item:
The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg.

In the procedure controls, the reference item was degraded by an average of 75% and 86% by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 93%.


Biodegradation in the Toxicity Control:
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the COD of the test item and the ThOD of the reference item.

In the toxicity control, the course of biodegradation over the 28 day exposure period was similar to the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation amounted to 58%.

Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 224 mg/L because biodegradation in the toxicity control was >25% within 14 days.

Chemical Oxygen Demand (COD) of the Test Item:
The chemical oxygen demand (COD) of the test item C.I. Leuco Sulphur Brown 96 was determined according to the EU Commission Directive 92/69/EEC, Part C.6 following DIN 38414-S9.

The duplicate values were 0.23 and 0.23 mg O2/mg test item. The mean COD was 0.23 mg O2/mg test item.


pH Measurement:
The pH measured in all flasks at the start of the test was in the range 7.4–7.5. At the end of exposure (Day 28), pH values of 7.0–7.5 were measured.


Validity of the Test:
The results are considered valid since the following criteria are met:

- The percentage biodegradation of the reference item sodium benzoate calculated from the oxygen consumption were higher than 40% of the ThOD after 7 days and higher than 65% of the ThOD after 14 days of incubation.

Key result

Parameter:

COD

Value:

0.23 g O2/g test mat.

Results with reference substance:

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg.

In the procedure controls, the reference item was degraded by an average of 75% and 86% by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 93%.

   Validity of the Test     

The results are considered valid since the following criteria are met:

 

The CO₂evolution in the inoculum controls at the end of the test was 28 and 33 mg/L (corresponding to 23 and 27 mg C per flask; criterion: 40 mg CO₂/L, corresponding to 33 mg C per flask).

The IC content in the test item flasks was 0% of the TC (criterion: less than 5% of the TC).

The difference of duplicate values for the degradation of the test item was less than 20% at the end of the test (criterion less than 20% at the plateau, at the end of the test or at the end of the 10‑day window

The percentage degradation of the reference item reached the level for ready biodegradability (>60% in a 10-day window) by Exposure Day 7 (criterion: at least 60% in a 10-day window by Day 14). 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item C.I. Leuco Sulphur Brown 96 was not biodegradable under the conditions of the test.

Executive summary:

The test item C.I. Leuco Sulphur Brown 96 was investigated for its inherent biodegradability in a manometric respirometry test over 28 days based on the OECD Guideline for Testing of Chemicals No  302 C (1981). The following modifications were made: activated sludge from one source was used, the activated sludge was not fed during the holding period of maximum seven days, the test was run at 22 °C, the test water composition was slightly changed, and only the biological oxygen demand (BOD) was monitored, no test item specific analysis was performed.

 

The oxygen consumption of the test item C.I. Leuco Sulphur Brown 96 in the test media was slightly lower than the normal range of the oxygen consumption in the inoculum controls.

 

Consequently, C.I. Leuco Sulphur Brown 96 was not biodegradable under the test conditions within 28 days.

 

In the toxicity control, containing both the test item and the reference item sodium benzoate,
C.I. Leuco Sulphur Brown 96 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 224 mg/L.

 

In the procedure controls, the reference item (sodium benzoate) was degraded by an average of 75% and 86% by Exposure Days 7 and 14, respectively, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 93%.

Description of key information

The ready and inherent biodegradability of the test substance was investigated according to the OECD guideline 301 B and OECD guideline 302 C. Under the test conditions, the test substance was found to be not biodegradable over the test period of 28 days. Furthermore, the test substance showed no inhibitory effect on the microorganisms.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

OECD 301B:

The test item C.I. Leuco Sulphur Brown 96 was investigated for its ready biodegradability in a 28‑Day CO2Evolution (Modified Sturm) Test according to the Method C.4-C of Commission Regulation (EC) No 440/2008, and the OECD Guideline for Testing of Chemicals, No. 301 B (1992) and the US EPA Draft Ecological Effects Test Guidelines OPPTS 835.3110 (Paragraph (m)).

The test item C.I. Leuco Sulphur Brown 96 was not biodegradable under the test conditions within 28 days.

In the toxicity control, containing both C.I. Leuco Sulphur Brown 96 and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, C.I. Leuco Sulphur Brown 96 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 167 mg/L.

In the procedure controls, average biodegradation of the reference item was 82% by Exposure Day 14, thus confirming suitability of the activated sludge (≥60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 94%.

OECD 302C:

The test item C.I. Leuco Sulphur Brown 96 was investigated for its inherent biodegradability in a manometric respirometry test over 28 days based on the OECD Guideline for Testing of Chemicals No  302 C (1981). The following modifications were made: activated sludge from one source was used, the activated sludge was not fed during the holding period of maximum seven days, the test was run at 22 °C, the test water composition was slightly changed, and only the biological oxygen demand (BOD) was monitored, no test item specific analysis was performed.

The oxygen consumption of the test item C.I. Leuco Sulphur Brown 96 in the test media was slightly lower than the normal range of the oxygen consumption in the inoculum controls.

Consequently, C.I. Leuco Sulphur Brown 96 was not biodegradable under the test conditions within 28 days.

In the toxicity control, containing both the test item and the reference item sodium benzoate, C.I. Leuco Sulphur Brown 96 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 224 mg/L.

In the procedure controls, the reference item (sodium benzoate) was degraded by an average of 75% and 86% by Exposure Days 7 and 14, respectively, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 93%.