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Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
october 1996
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Constituent 1
Reference substance name:
Cas Number:
Molecular formula:
C30 H63 Cl N2 O3
Test material form:
aqueous solution
Details on test material:
Lot# D-425-156
clear amber liquid
Specific details on test material used for the study:
storage conditions: room temperature, 10 - 30C in darkness

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
a sample of activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (Yorkshire water), a treatment works whose waste-water catchment is predominantly domestic. On arrival in the laboratory, the sample was aerated by means of a compressed air supply delivered through a diffuser block.
The suspended solids concentration was determined by filtering a 25 mL subsample through a pre-dried and pre-weighed glass microfibre filter. The filter and retained solids were then dried by microwave oven, re-weighed and the contribution made by the sludge solids determined by difference.

The activated sludge inoculum was not acclimatised or adapted to the test material before exposure to the the test substance in this study
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
16.2 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Confirmatory TOC analysis
A secondary stock was made from the primary by diluting 20 mL of the latter in 1L with reverse-osmosis water. The secondary stock, whose TOC concentration was nominally 45 mg C/L was subjected to confirmatory analysis, performed in triplicate on duplicate samples by means of a Rosemount Dihrmann DC 190 TOC analyser.
For the purpose of this confirmatory analysis, the analyser was run in the automatic mode and calibrated against a 50 mg C/L potassium hydrogen phthalate standard.
The mean result of this analysis was 45.43 mg C/L which compared satisfactorily with the nomianl TOC concentration of the secondary stock. The analysis confirmed that the primary stock solution provided a suitable vehicle for adding the required quantity of sodium benzoate to the reference toxicity control vessels.

Test medium
The test was conducted in a synthetic mineral salts medium based on reverse-osmosis water. See study report for coposition of the medium.
On the basis of the suspended solids determination described above, the medium concentrate was inoculated with activated sludge to provide a nominal solids concentration of 90 mg/L. The medium was then dispersed at the rate of 1L per test vessel and these volumes made up to 3L by addition of reverse-osmosis water. The final suspended solids concentration in all vessels was thus nominally 30 mg/L

Dosing the test substance
The test substance was dosed to the duplicate test vessels and the toxicity control by adding to each a 0.24 mL volume of the test substance concentrate. In all cases, these aliquots were expected to give the test material concentrations corresponding to 15 mg C/L. However, after correcting for the percentage carbon content obtained by elemental micro-analysis, and after adjusting for density, the dose concentrations were nominally 16.2 mg C/L. The implications of hte difference are discussed in appendix 4 of the study report.

Dosing the reference substance
the reference substance was dosed to the duplicate reference vessels and the toxicity control by delivering to each a 20 mL volume of the primary stock solution. In all three cases, these aliquots gave a nominal sodium benzoate concentration corresponding to 15 mg C/L

Environmental control
Temperature recorded durng the study wre in the range 20.3 to 22.3C
pH measurements made in all vessels at the start and end of incubation. Final pH were made on day 28 immediatly before vessels were acidified to release any residual CO2 remaining in solution.

air flow was regulated too. See study report for more details
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
See the atached study report for details on results

BOD5 / COD results

Results with reference substance:
Final degradations values of 70 and 73 % and at no time did degradation in either replicate diverge from that recorded in the other one by more than 6%.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
inherently biodegradable
CO2 evolution from the substance did not exceed 38% of theoretical at the applied concentration over the sourse of the 28 day incubation. The substance cannot therefore be classified as readily biodegradable. Nevertheless, the results of this study show that the substance is inherently biodegradable.
Executive summary:

The ready biodegradability of the substance was assessed according to CO2 evolution test. The method used was that described in Part C4-C of EC commission Directive 92/69/EEC and in the 1992 revision of OECD Guideline 301B.

CO2 evolution from the substance did not exceed 38% of theoretical at the applied concentration over the sourse of the 28 day incubation. The substance cannot therefore be classified as readily biodegradable. Nevertheless, the results of this study show that the substance is inherently biodegradable.

The ready biodegradability methods were advised, not as simuations of realistic aquatic environment, but as stringent fail-safe tests to screen for and to classify substances that would degrade rapidly and completely in natural water bodies. The failure of the test material to undergo complete mineralisation under stringent conditions employed in this study is not necessarily an indication that the substance is not fully biodegradable.