reaction mass of cis 4-(3-methylbutyl)cyclohexanol and trans 4-(3-methylbutyl)cyclohexanol

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:

June 15, 2007 - August 14, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

, 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2007-03-21

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Symrose
- Substance type: technical product
- Physical state: colourless, liquid
- Storage condition of test material: ambient temperature, dark and dry

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Fresh samples of activated sludge are withdrawn on July 5th, 2007 from the sewage treatment plant Ruhrverband Kläranlage, Schmallenberg, Germany, which is mainly fed with municipal wastewater. The samples were washed once with mineral medium after the arrival at the laboratory and kept aerobic until use. Preparation of inoculum for exposure: aeration; Concentration of sludge: The concentration used in the test was 29.6 mg dry mass/litre (7.40 mg dry mass/250 mL).

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Mineral medium
For the preparation of the stock solutions for mineral medium the following reagents (analytical grade) were used :
( a ) KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4 x 2 H2O 33.40 g/L; NH4Cl 0.50 g/L
( b ) CaCl2 x 2 H2O 36.40 g/L
( c ) MgSO4 x 7 H2O 22.50 g/L
( d ) FeCl3 x 6 H2O 0.25 g/L.
The mineral medium applied in the test contained 10 mL/L and 1 mL/L of the mineral stock solution a and b–d, respectively.
- Test temperature: 22°C
- pH: Before test start the pH values of all solutions were determined and found to be 7.4 ± 0.2. At the end of the test the pH values of all solutions were determined again.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Symrose and sodium benzoate, respectively, in concentrations of about 100 mg/L mineral medium were incubated with 29.6 mg dry mass inoculum/L mineral medium in 500 mL glass vessels at a medium volume of 250 mL.
- Number of culture flasks/concentration: Test vessels:
Test suspension: 2 vessels containing test item and inoculum
Inoculum blank: 2 vessels containing only inoculum
Abiotic control: 2 vessels containing test item and a sterilising agent
Procedural control: 2 vessels containing reference item and inoculum
Toxicity control: 2 vessels containing test item, reference item and inoculum
- Method used to create aerobic conditions: The suspension was aerated during the whole test.
- Measuring equipment: The measurement and recording of the oxygen demand was carried out continuously using a SAPROMAT respirometer (VOITH Inc.).
- Treatment of results: The Theoretical Oxygen Demand was calculated on the basis of the sum formula of the test and reference item by ThOD [g/g] = 16 * (2 C + 1/2 H + 1/2 Na – O) / Molecular weight. The ThOD values for Na-benzoate, the test item and the toxicity control were determined as follows:
ThOD(Symrose): 3.01 mg O2/mg test item
ThOD(Na-Benzoate): 1.67 mg O2/mg reference item
ThOD(toxicity control): 2.34 mg O2/mg substance mixture
The Biochemical Oxygen Demand (BOD) was calculated on the basis of the test raw data by BOD [mg/mg] = mg O2 uptake corrected by blank per mg test item. The percent degradation was calculated according to the following formula:
Dt = [(Ct –Cb) / ThOD] x 100
Dt: degradation (%) at time t;
Ct: mean oxygen consumption (mg/L) in the test suspension at time t;
Cb: mean oxygen consumption (mg/L) in the blanks at time t;
ThOD: Theoretical oxygen demand of the test suspension (mg/L).

No further details are given.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

The concentration in the test vessels with reference item (procedural control, toxicity control) was 100 mg/L mineral test medium (25 mg/250 mL).

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

The biodegradation of Symrose in the static test was found to be 0 % after 28 days. Thus, no biodegradation within a 10-day-window could be obtained and no lag phase / adaptation phase was noticeable.
No abiotic degradation of Symrose was noticeable after 28 days of incubation.
Due to a biodegradation below 60 % within the test duration and thus within a 10-day window, the test item Symrose must be identified as not readily biodegradable under the chosen test conditions.

BOD5 / COD results

Results with reference substance:

The biodegradation of the item mixture in the toxicity control was found to be 31 % after 14 days of incubation. Thus, the demanded threshold value of 25 % is exceeded and the test item Symrose can be identified as non toxic in a ready biodegradability test.
The reference item sodium benzoate was degraded to 81 % within the first 14 days.

Any other information on results incl. tables

Table 1: Oxygen consumption. Cumulated consumption (mg/O2/L) after 14 days of incubation. Single and mean values of the parallel test vessels and standard deviation. Test suspension and procedural control: 100 mg/L; Toxicity control 200 mg/L

Vessel	Test suspension	Inoculum blank	Abiotic control	Precedural control	Toxicity control
1	16	18	0	153	169
2	13	20	0	156	160
Mean	15	19	0	155	165
SD	2	1	0	2	6

Table 2: Percent degradation. Degradation (%) after 14 days of incubation. Single and mean values of the parallel test vessels and standard deviation.

Vessel	Test suspension	Abiotic control	Procedural control	Toxicity control
1	-1.0	0	80.5	32.1
2	-2.0	0	82.3	30.2
Mean	-1.5	0	81.4	31.1
SD	0.7	0	1.3	1.4

Table 3: Oxygen consumption. Cumulated consumption (mg/O2/L) after 28 days of incubation. Single and mean values of the parallel test vessels and standard deviation.

Vessel	Test suspension	Inoculum blank	Abiotic control	Precedural control	Toxicity control
1	23	23	1	159	189
2	17	24	1	174	166
Mean	20	24	1	167	178
SD	4	1	0	11	16

Table 4: Percent degradation. Degradation (%) after 28 days of incubation. Single and mean values of the parallel test vessels and standard deviation.

Vessel	Test suspension	Abiotic control	Procedural control	Toxicity control
1	-0.2	0.3	81.4	35.4
2	-2.2	0.3	90.4	30.5
Mean	-1.2	0.3	85.9	33.0
SD	1.4	0.0	6.4	3.5

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The biodegradation of Symrose in the static test was found to be 0 % after 28 days. Thus, no biodegradation within a 10-day-window could be obtained and no lag phase / adaptation phase was noticeable.
No abiotic degradation of Symrose was noticeable after 28 days of incubation.
The biodegradation of the item mixture in the toxicity control was found to be 31 % after 14 days of incubation. Thus, the demanded threshold value of 25 % is exceeded and the test item Symrose can be identified as non toxic in a ready biodegradability test.
The reference item sodium benzoate was degraded to 81 % within the first 14 days.
Due to a biodegradation below 60 % within the test duration and thus within a 10-day-window, the test item Symrose must be identified as not readily biodegradable under the chosen test conditions.

Executive summary:

The test is considered valid, as

-    the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20 %,

-    the percentage degradation of the reference item has exceeded the pass level of 60 % by day 14,

- the oxygen uptake of the inoculum blank is < 60 mg/L in 28 days and the pH value was inside the range of 6.0 - 8.5.