2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

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:

1993-11-08 - 1993-12-08

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to directive 88/320 EEC, date of inspection: 1992-03-17

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Type: A mixed population of activated sludge microorganisms
Source: The aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
Date of collection: 8 November 1993.
Pre-Treatment: The activated sludge was homogenised for approximately 2 minutes and allowed to settle for 1/2 hour. The supernatant was centrifuged to remove coarse solids and used for testing.
Usage rate: 1% inoculum.

Duration of test (contact time):

28

Initial conc.:

33.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test sample:
Method of Preparation: Direct dispersion of the test substance in culture medium

Exposure
Duration: 28 days.
Vessels: 5 litre glass culture vessels containing 3 litres.
Culture medium: As specified in OECD-guideline 301
Loading: 1% inoculum per test vessel.
Test concentration: 20 mg carbon/L in duplicate.
Reference material 10 mg carbon/L sodium benzoate, in duplicate.
Test series:
1. Culture medium with inoculum in duplicate.
2. Culture medium with inoculum and sodium benzoate in duplicate
3. Culture medium with inoculum and test substance in duplicate.
4. Culture medium with inoculum and test substance, poisoned by the addition of 10 ml of a 10 g/1 mercuric chloride solution to act as an abiotic control (one vessel only).

Temperature: 21 ± 1°C
Light regime: The study was carried out in darkness.
Procedure: Approximately 24 hours prior to the start of the study the vessels were filled with 2400 mL of culture medium and 30 mL of inoculum and aerated overnight. On day 0 the test and standard materials were added and the volume in all vessels made up to 3 litres by the addition of culture medium. The CO2 absorption bottles were connected to the outlet of the vessels on day 0.

Aeration: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/min and stirred continuously by magnetic stirrers. CO2-free air was produced by sparging compressed air through the following series:
- 3 x 500 ml Dreschel bottles filled with 350 mL 10 N NaOH.
- 1 x 500 ml Dreschel bottle filled with 350 mL 0.025 N Ba(OH)2
- 1 x 500 ml empty Dreschel bottle to prevent liquid carry-over.

CO2-absorption: 2 x 500 mL Dreschel bottles filled with 350 mL 0.05 M NaOH. All CO2 absorbing solutions were prepared using purified de-gassed water.

Sampling: Samples (2 mL) were taken from the first CO2 absorber vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29.
On day 28 the pH of each test vessel was measured and 1 mL of concentrated HCl added to drive off inorganic carbonate. The vessels were aerated overnight and the final samples were taken from both absorber vessels on day 29.

Apparatus: Ionics 555 TOC Anlyser. Each analysis was carried out in triplicate.

Evaluation: All calculations were performed according to the guideline (OECD 301B).

Reference substance:

benzoic acid, sodium salt

Preliminary study:

not applicable

Test performance:

not applicable

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

28 d

Details on results:

The pH values of the test material, standard material and control cultures an day 28 were 7.5 and 7.5 (test material), 7.5 and 7.5 (standard material), 7.5 and 7.5 (control) and 7.4 (abiotic control).
The test substance attained 1% degradation after 28 days and so, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.
Additional investigational work was carried out using the Activated Sludge Respiration Inhibition test method (OECD Guideline No. 209) which showed that the test substance did not inhibit the respiration of sewage sludge microorganisms at the test concentration employed in the test. In the light of this the inhibition control vessel (test substance plus sodium benzoate) was omitted from the test.
Therefore from these results it is evident that the test substance is not readily biodegradable and that this result is not due to a toxic effect on the inoculum.

Results with reference substance:

Sodium benzoate attained 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

A ready biodegradability test on the test substance according to OECD 301B (CO2 -evolution) and further testing for activated sludge respiration inhibition (OECD 209, results only mentioned in the study report) it is evident that the test substance is not readily biodegradable (1% biodegradation) and that this result is not due to a toxic effect on the inoculum.
Sodium benzoate attained 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Executive summary:

In a ready biodegradablility test (reliability categoriy 1) on the test substance according to OECD 301B (CO2 -evolution) and complient to GLP, the test substance attained 1% degradation at a concentration of 20 mg C/L. To exclude toxicity of the test item to the inoculum at the tested concentration additional investigational work was carried out using the Activated Sludge Respiration Inhibition test method (OECD Guideline No. 209, results mentioned in this report, but no study report available) which showed that the test substance did not inhibit the respiration of sewage sludge microorganisms at the test concentration employed in the test. Therefore from these results it is evident that the test substance is not readily biodegradable and that this result is not due to a toxic effect on the inoculum.

Sodium benzoate attained 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995-02-22 - 1995-03-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study followed relevant guidelines and is compliant to GLP. Additionally, analytical tests and toxicity tests not required by the guideline had been performed. The results are well documented and plausible. However, sampling of activated sludge should have been performed from 10 different sites (only one) and variability between replicates of test samples was too high as to fulfill this validity criterium (25% instead of <20%).

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes

Remarks:

according to OECD codes of GLP, May 1981, Doc C (81)30 (Final)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The inoculum was activated sludge which was obtained from the sewage treatment plant in Usserod. The influents to the waste water plant are dominated by municipal waste water. The sludge was collected an February 22, 1995, and was diluted with the test medium to achieve a final concentration of suspended solids corresponding to 30.0 mg SS/L (d.w).

Duration of test (contact time):

28 d

Initial conc.:

50 mg/L

Based on:

other: Total Carbon content

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

other: Quantification of the dry weight of suspended solids (pigment coponent) at start (3 hours of incubation) and termination of test.

Parameter followed for biodegradation estimation:

other: HPLC determination of possible degradation products (t0, t28).

Details on study design:

Based an the carbon content of the test product (0.33 mg C/mg), 153.3 mg of product per litre giving a final concentration of 50 mg C/L was added from an aqueous stock solution (1.01 g/L) into the required volume of test medium. The carbon content of the product was verified by chemical analysis. The theoretical oxygen demand (ThOD) for a complete mineralization of the test product was quantified by measuring the chemical oxygen demand (COD, potassium dichromate method).
Sodium benzoate was used as the reference substance, and the test concentration of 100 mg/L was achieved by addition of 10 mL per litre from an aqueous stock solution containing 10.0 g/L. The medium, to which either test product, reference substance, or both were added, was dispensed into the test vessels.
The test medium (mineral medium) was prepared as described in the guideline.

The following test vessels were prepared:
- 3 containing inoculum alone (inoculum control)
- 3 containing test product and inoculum (test suspensions)
- 3 containing reference substance and inoculum (activity control)
- 3 containing test product, reference compound and inoculum (inhibition control)

The various additions to the test vessels were as follows:
Vessel 1-3: 415 mL of inoculated medium
Vessel 4-6: 415 mL of inoculated medium with test product
Vessel 7-9: 275 mL of inoculated medium with reference substance
Vessel 10-12: 275 mL of inoculated medium with test product and reference substance
The test vessels were incubated in the dark at 20 ± 1 °C with magnetic stirring.
The produced carbon dioxide was trapped in an absorber with KOH which was placed inside the closed vessels. The oxygen consumption was followed by frequent recordings of the pressure reduction in the BOD-meters.
Calculations/evaluations were performed according to the guideline.

Toxicity assay:
The Microtox test was used to study the possible changes in the toxicity of the test product resulting from biodegradation processes. Microtox is a commercially available toxicity assay using the light emission from the marine bacterium Vibrio fischeri as test parameter. This assay is suitable for routine screening purposes. The toxicity of the test item at the applied test concentration was examined with subsamples of the test suspensions after 3 hours (to) and at the termination of the 28-day period (t28).

Reference substance:

benzoic acid, sodium salt

Preliminary study:

not applicable

Test performance:

not applicable

Key result

Parameter:

% degradation (O2 consumption)

Value:

16

St. dev.:

5.29

Sampling time:

28 d

Remarks on result:

other: % of ThOD

Key result

Parameter:

% degradation (TOC removal)

Value:

24

Sampling time:

28 d

Details on results:

The results of the dry weight quantification of the precipitated pigment in the test vessels are shown in Table 1.  Although the figures in Table 1 are very coarse estimates, they indicate that degradation of test item was negligible.
During the 28-day period the oxygen consumption (BOD) from the test item reached a level corresponding to 16% of the theoretical oxygen demand (ThOD) for a complete mineralization of the product. Approximately 70% of the total carbon in the product is contained in the pigment (equal to submission substance). Assuming that the pigment component was stable, the observed BOD of 16% indicates that, besides carbon assimilation by the microorganisms, approximately 53% of the additives were mineralized during the test. Also the HPLC-analysis of the pigment component and three possible degradation products inidcated stability of the pigment during the test.
A comparison between the oxygen consumption, when the test item and sodium benzoate were added to separate test vessels or in combination, showed that the product did not inhibit the respiratory activity in the sludge at the applied concentration.
The oxygen uptake in the inoculum control (without test product) was 15 mg O2/L in 28 days.
Toxicity assay (Microtox test):
The Microtox test showed that the applied concentration of the test item did not inhibit the light emission from Vibrio fischeri at the start of the test (t0, i.e. after 3 hours) or after incubation for 28 days. No effect was observed at the highest examined concentration which was 500 mL of test suspension per litre. This indicates that metabolites with a higher toxicity than the original ingredients were not produced.

Results with reference substance:

The activated sludge had a satisfactory activity as illustrated by the degradation of the reference compound. The oxygen consumption from sodium benzoate corresponded to 80% of the theoretical oxygen demand after 7 days, and 91% of this compound was degraded at the termination of the test.

Table I. Estimation of precipitated pigment in test suspensions.

Sampling time	Test vessels with product (mg SS dw/L)	Blank; no product (mg SS dw/L)	Net weight esti- mate for pigment precipitate (mg SS dw/L)
to(3 h)	87.2	45.9	41.3
t28	97.3	44.9	52.4

Validity criteria fulfilled:

yes

Remarks:

Two of the validity criteria prescribed in the test guideline were fulfilled: (1) The oxygen uptake in the controls was below 60 mg O2/L. (2) The degradation of the reference substance as calculated from oxygen consumption exceeded 40% after 7 days and 65

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

A test on ready biodegradability had been performed on the test item, a water based pigment dispersion with approximately 70% of the contained organic carbon derived from pigment, which is identical to the submission substance. Therefore, the test results are considered adequate to fulifl the endpoint requriements.
Under the test condiitions (according to relevant guidelines, compliant to GLP, reliability 2), no biodegradation had been observed with respect to the pigment portion. 16% of the theoretical oxygen demand for complete mineralization had been found for the test item, suggesting approximately 53% of the product additives contained besides the submission substance being mineralized during 28 days.
No toxicity of the test item could be observed in terms of inhibition of activated sludge activity in tests with the referenc substance (sodium benzoate) compared to tests with the reference substance alone. No toxicity of the test suspension could be observed neither at the beginning of the test nor at the termination (28 d) as judged from Microtox assays performed with 500 mL of test suspenision per litre.
Such, the submission substance proved to be not biodegradable during the test period of 28 days and conditions of OECD 301C. However, it proved to be not at all inhibitory to activated sludge and non-toxic to Vibrio fischeri in the Microtox test at day 0 and day 28, respectively.

Executive summary:

The biological oxygen demand from degradation of the test item was 16% of the theoretical oxygen demand for a complete mineralization of the product. Analyses of the pigment component and of three possible degradation products (3,3'-dichlorobenzidine,2HCl, 2, 4-dimethylacetanilid and dimethylaniline) indicated that the pigment was stable during the test. Assuming complete persistency of C.I. Pigment Yellow, the BOD measurements suggest that approximately 53% of the product additives were rnineralized during 28 days.

The activated sludge had a satisfactory activity as more than 80% of the reference substance was degraded within the first two weeks of the test period. The applied concentration of the test product did not inhibit the respiratory activity of the inoculum.

The toxicity assay, using the Microtox system, indicated that degradation of the test item did not lead to the formation of degradation products with a higher toxicity than the original ingredients.

Two of the validity criteria prescribed in the test guideline were fulfilled:

- the oxygen uptake in the controls was below 60 mg O₂/L

-  the degradation of the reference substance as calculated from oxygen consumption exceeded 40% after 7 days and 65% after 14 days.

However, The difference of extremes of the replicate BOD values exceeded 20%. The observed variation is not uncommon in test runs with relatively low BOD-values.

Description of key information

All available tests performed with structural analogues, the Diarylide Yellow Pigments, point to low biodegradability. Therefore, the substances are to be regarded as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater