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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:
key study
Study period:
May-June 2001
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted under GLP conditions and in accord. with EEC directive 92-69 and OECD guideline No. 301 B.

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
Chemical structure
Reference substance name:
(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid
EC Number:
EC Name:
(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid
Cas Number:
Molecular formula:
(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid
Details on test material:
- Description : white powder
- Purity : 98,9 %
- Test substance storage : in refrigerator in the dark
- Stability under storage conditions : stable
- Expiry date : 3 March 2002

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.

- Treatment
The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 5.0 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle {30-90 minutes) and the liquid decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Duration of test (contact time):
ca. 28 d
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Test duration : 28 days {last C02-measurement on the 29th day).

- Test vessels : 2 litre all-glass brown coloured bottles.

- Milli-RO I Milli-Q water : Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion­ exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

- Stock solutions of mineral components
A) 8.50 g KH2P04
21.75 g K2HP04
67.20 g Na2HP04.12H20
0.50 g NH4CI
dissolved in 1 I Milli-Q water, pH 7.4 ± 0.2
B) 22.50 g MgS04.7H20 dissolved in 1 I Milli-Q water.
C) 36.40 g CaCI2.2H20 dissolved in 1 I Milli-Q water.
D) 0.25 g FeCI3.6H20 dissolved in 1 I Milli-Q water.

- Mineral medium : 1 I mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

- Barium hydroxide : 0.0125 M, stored in a sealed vessel to prevent absorption of C02 from the air.

- CO2 : A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap C02 which might be present in small amounts. The C02-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min), except for a temporary breakdown in the aeration (< 1 day) on day 16. However, thisbreakdown was considered to have no effect on the outcome of this study.

- Test concentration
The test substance was tested in duplicate at 50 mg per 2 litres, corresponding to 12 mg TOC/l. The organic carbon content was based on the
molecular formula.

- Preparation of bottles:
* Pre-incubation medium :
Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with C0 2-free air overnight to purge the system of C02.
* Type and number of bottles :
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance (ca.40 mg/l sodium acetate (Merck art. 1062680250, batch TA 820068 033), TOG= 12 mg/l) and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
* Preparation
The test substance and positive control were added to the bottles.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
Three C02-absorbers (bottles filled with 100 ml 0.0125M Ba(OH)2) were connected in series to the exit air line of each test bottle.
* Start of the incubation
The test was started by bubbling C02-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
29 d

Any other information on results incl. tables

 Day  Biodegradation (%) Mean A and B
 0  0
 2  13
 5  41
 7  53
 9  60
 12  68
 14  73
 19  76
 23  77
 27  80
 29  80

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Shikimic acid was readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

SHIKIMIC ACID was tested for its ready biodegradability in the carbon dioxide (C02) evolution test (modified Sturm test) at 50 mg per 2 litres, corresponding to 12 mg TOC/l.

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

The Theoretical C02 production (ThC02) of SHIKIMIC ACID was calculated to be 1.77 mg C02/mg.

Since SHIKIMIC ACID is well soluble in water, the test media were prepared using a stock solution of 1 g/l in Milli-RO water. The test solutions were continuously stirred during the test.

The relative degradation values calculated from the measurements performed during the test period revealed significant degradation of SHIKIMIC ACID, i.e. 76 and 85% for A and B respectively. Furthermore, biodegradation of SHIKIMIC ACID of at least 60% was reached within 10 days of biodegradation exceeding 10%.

In the toxicity control SHIKIMIC ACID was found to be not inhibitory.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, SHIKIMIC ACID was readily biodegradable under the conditions of the modified Sturm test presently performed.