2,2'-[(2,2',5,5'-tetrachloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide]

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key 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%).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

225-822-9

EC Name:

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

Cas Number:

5102-83-0

Molecular formula:

C36H34Cl2N6O4

IUPAC Name:

2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[N-(2,4-dimethylphenyl)-3-oxobutanamide]

Specific details on test material used for the study:

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

Study design

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 test substance concentration

Initial conc.:

50 mg/L

Based on:

other: Total Carbon content

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation 3

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 4

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

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

not applicable

Test performance:

not applicable

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

16

St. dev.:

5.29

Sampling time:

28 d

Remarks on result:

other: % of ThOD

% Degradation 2

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.

BOD5 / COD results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.