Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was conducted between 25 October 2002 and 02 December 2002.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot No.: 9000477976
Purity: 97.4% (sum of the four isomers)
Aspect: colourless to pale yellow liquid
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Peney-Dessous) was used. The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
Dry weight of suspended solids: 3.970 g/I
To obtain a concentration of 30 mg/I (dry weight) in a 250 ml flask, 1.89 ml of sludge is needed (inoculum).
Duration of test (contact time):
35 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Water
The water used during this study is deionised water containing less than 10 mg/L dissolved organic carbon.

Stock solutions of mineral components
The following stock solutions were prepared:

Solution A:
KH2PO4: 8.5 g
K2HPO4: 21.75 g
Na2H PO4.2H2O: 33.4 g
NH4CI: 0.5 g
dissolved in water and made up to 1 litre.

Solution B:
CaCl2: 27.5g
dissolved in water and made up to 1 litre.

Solution C:
MgSO4•7H2O: 22.5g
dissolved in water and made up to 1 litre.

Solution D:
FeCI3.6H2O: 0.25g
HCI Conc.: one drop
dissolved in water and made up to 1 litre .

Mineral medium
Prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of the solutions B, C and D and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.

Toxicity of the test substance
As suggested in the OECD 301F method, the toxicity of the test substance for the inoculums is checked. Therefore a pair of flasks of the volumetric respirometer are filled with mineral medium + test substance (100 mg/L) + reference substance (100 mg/L) + inoculums and their respirations are recorded as for the other flasks. If they are lower than those of the flasks containing mineral medium + reference substance (100 mg/L) + inoculums, the test substance can be assumed to be inhibitory to the inoculums used.

Determination of the dry weight of suspended solids
The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 – 110 °c for two hours and weighing the residue.

Preparation of the flasks
Test substance samples (25 mg, corresponding to 100 mg/l in a 250 ml flask) are weighed in small aluminium boats and added directly to the test flasks of the SAPROMAT, whereas reference substance samples (sodium benzoate) are added as 1.0 ml of a 25 mg/ml solution in mineral medium.
All flasks are filled with 250 ml of mineral medium. Samples of test or reference substance, or both, are added. Then, a volume of suspended sludge corresponding to 7.5 mg dry weight (as determined, generally 1 to 3 ml) is added. Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. About 2 g of soda lime is placed in an attachment of the stopper, the flasks are closed and placed in the water bath of the SAPROMAT. After temperature and pressure equilibration, the oxygen-meters of the instrument are set to zero (time zero of the experiment).

Performance of the test
Every day the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period (normally 28 days), the pH of each flask is measured again.

Test temperature: 22°C
Test duration: 35 days
Reference substance:
benzoic acid, sodium salt
Remarks:
100 mg/L
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
35 d
Details on results:
Oxygen uptakes, as read on the SAPROMAT meters, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.
Degradation of sodium benzoate exceeds 40 % after 7 days and 65 % after 14 days: the activity of the inoculum is thus verified (validity criterion).
Oxyoctaline formate undergoes no biodegradation after 35 days in the test conditions. The curves obtained with the reference substance alone and with Oxyoctaline formate + reference substance show no toxic effect of Oxyoctaline formate to the micro-organisms at the test concentration (100 mg/I). Thus, Oxyoctaline formate should be regarded as not readily biodegradable according to this test.

Biodegradability - Respirometric method - Test substance

Theoretical oxygen demand

Formula: C15H24N0O2

Molecular weight: 236.35

ThOD: 2.71 mg O2/mg

Biological oxygen demand (BOD, mg O2/L, adjusted to nominal concentrations:

 

Flask

 

Days:

5

7

14

21

28

35

BOD sludge

1st

B1

15.0

20.0

25.0

29.0

31.0

33.0

2nd

B2

17.0

20.0

26.0

34.0

40.0

48.0

mean

B

16.0

20.0

25.5

31.5

35.5

40.5

BOD Test Substance

1st

C1

12.2

15.3

19.4

24.4

26.6

31.6

2nd

C2

12.1

15.1

19.1

25.1

28.2

32.2

1stcorrected

C1-B

-3.8

-4.7

-6.1

-7.1

-8.9

-8.9

2ndcorrected

C2-B

-3.9

-4.9

-6.4

-6.4

-7.3

-8.3

% degradation

1st

D1

-1

-2

-2

-3

-3

-3

2nd

D2

-1

-2

-2

-2

-3

-3

mean

D

-1

-2

-2

-2

-3

-3

B = (B1 + B2)/2

D1 = 100 * (C1 - B)/ThOD * [S]

D2 = 100 * (C2 - B)/ThOD * [S]

D = (D1 + D2)/2

[S] = initial test substance concentration (mg/L)

Biodegradability - Respirometric method - Sodium benzoate

Theoretic al oxygen demand

Formula: C7H5O2Na1

Molecular weight: 144.1 g/mol

ThOD: 1.67 mg O2/mg

Biological Oxygen Demand (BOD, mg O2/L, adjusted to nominal concentrations):

 

Days

5

7

14

21

28

35

BOD Sludge

1stflask

B1

15.0

20.0

25.0

29.0

31.0

33.0

2ndflask

B2

17.0

20.0

26.0

34.0

40.0

48.0

Mean

B

16.0

20.0

25.5

31.5

35.5

40.5

BOD Refer. Subst.

1stflask

A1

131.8

151.8

173.8

189.9

197.8

203.8

2ndflask

A2

129.9

141.8

157.8

163.8

165.8

167.8

1stfl. Corr.

A1-B

115.8

131.8

148.3

158.3

162.3

163.3

2ndfl. Corr.

A2-B

113.9

121.8

132.3

132.3

130.3

127.3

% Degr.

1stflask

D1

70

79

89

95

97

98

2ndflask

D2

68

73

79

79

78

76

Mean

D

69

76

84

87

88

87

B = (B1 + B2)/2

D1 = 100 * (A1 - B_/ThOD * [S]

D2 = 100 * (A2 - B)/ThOD * [S]

D = (D1 + D2) / 2

[S] = Initial reference substance concentration (mg/L).

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Oxyoctaline formate undergoes no biodegradation after 35 days in the test conditions. The curves obtained with the reference substance alone and with Oxyoctaline formate + reference substance show no toxic effect of Oxyoctaline formate to the micro-organisms at the test concentration (100 mg/I). Thus, Oxyoctaline formate should be regarded as not readily biodegradable according to this test.
Executive summary:

The ready biodegradability of Oxyoctaline formate was determined by Manometric Respirometry test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F.

Oxyoctaline formate undergoes no biodegradation after 35 days in the test conditions.

The curves obtained with the reference substance alone and with Oxyoctaline formate + reference substance show no toxic effects of Oxyoctaline formate to the microorganims at the test concentration (100 mg/L).

Thus, Oxyoctaline formate should be regarded as not readily biodegradatble according to this test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 20 October 2015 and 16 February 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
An optional toxicity control as suggested by OECD guideline 301F was not performed. The validity of the study is not adversely affected.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot No.: 9000477976
Purity: 96.3% (sum of four isomers)
Aspect: colourless to pale yellow liquid
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-bay, Satigny, Switzerland) was used. The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
Dry weight of suspended solids: 3.9027 g/I, diluted to 2.62 g/l
To obtain a concentration of 30 mg/I (dry weight) in 437 ml total volume, 5ml of sludge (inoculum) was added to 432ml of mineral medium.
Duration of test (contact time):
42 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Water
The water used during this study is ultrapure water, containing less than 5 ppb total organic carbon, produced by using a Millipore Direct-Q 3 UV purification system

Stock solutions of mineral components
The following stock solutions were prepared:

Solution A:
KH2PO4: 8.5 g
K2HPO4: 21.75 g
Na2H PO4.2H2O: 33.4 g
NH4CI: 0.5 g
dissolved in water and made up to 1 litre.

Solution B:
CaCl2: 27.5g
dissolved in water and made up to 1 litre.

Solution C:
MgSO4•7H2O: 22.5g
dissolved in water and made up to 1 litre.

Solution D:
FeCI3.6H2O: 0.25g
HCI Conc.: one drop
dissolved in water and made up to 1 litre .

Mineral medium
Prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of the solutions B, C and D and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.

Determination of the dry weight of suspended solids
The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 – 110 °c for two hours and weighing the residue.

Preparation of the flasks
Test substance samples (13.1 mg, corresponding to 30 mg/l in 437 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop. For reference substance samples 43.7mg (corresponding to 100mg/l in 437 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop.
Flasks are filled with437 ml of mineral medium. Samples of test or reference substance are added. Then, 5ml of suspended sludge diluted to a concentration of 2.62 g/l dry matter is added. Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. Two sodium hydroxide pellets are placed in the quivers on top of the bottle and the flasks are closed tightly with the measuring heads. The flasks are allowed to equilibrate to the test temperature. The measurement is started by programming the measuring unit of the Oxitop flaks, and the test flask are plaved in the termperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

Performance of the test
Every day the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period (normally 28 days), the pH of each flask is measured.

Test temperature: 21.3 - 21.8°C
Test duration: 42 days
Reference substance:
benzoic acid, sodium salt
Remarks:
100 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
42 d
Parameter:
% degradation (test mat. analysis)
Value:
55.5
Sampling time:
42 d
Remarks on result:
other: replicate 1, based on 44.5% of initial nominal concentration found
Parameter:
% degradation (test mat. analysis)
Value:
57.6
Sampling time:
42 d
Remarks on result:
other: replicate 2, based on 42.4% of the initial nominal concentration found
Details on results:
- Oxygen uptakes, as read on the Oxitop controller, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.
- Degradation of sodium benzoate exceeded 40 % after 7 days and 65 % after 14 days: the activity of the inoculumwas thus verified (validity criterion).
- The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled. Therefore, the test is considered valid.
- Oxyoctaline formate undergoes, based on the oxygen consumption observed, no biodegradation after 28 days in the test conditions. Thus, Oxyoctaline formate should be regarded as not readily biodegradable according to this test.
- At the test concentration Oxyoctaline Formate did inhibit the intrinsic respiration of the inoculum by more than 20% on several days of the test. For example, on day 14 the O2 uptake in the test flask was only 16.5 mg O2/l compared to 22.3 mg O2/l in the inoculum blank (see Appendix 3 in study report, table 1 below). The inhibition effect is also clearly seen in the time plot, where negative biodegradation levels are observed on most days (appendix 2 in study report). Slight toxicity towards the inoculum at test concentration can therefore not be excluded.
- In a non-GLP part of the study the content of the test substance flask was analysed using a specific analysis (GC-FID and GC-MS) at the end of the test (day 42).
- Quantification of the residual Oxyoctaline formate (peak at retention time of 8.22 minutes on the GC-FID chromatograms) in the two replicate flasks showed 44.5% and 42.4% of the initial nominal concentration, respectively.
- A major degradation product (peak at a retention time of 7.77 min on the GC-FID chromatograms) was observed in both flasks which was tentatively identified as 1,3,6,7-tetramethylbicyclo(4.4.0)dec-7-ene-2-ol (= Oxyoctaline alcohol) using GC-MS analysis and comparison with library spectra.
- Using the relative peak areas on GC-FID chromatograms relative quantities of this degradation product were estimated to be 24.1% and 31.4% of the initial nominal concentration of oxyoctaline formate, on the two replicate flask at test end respectively.

Biodegradability - Respirometric method - Test substance

Theoretical oxygen demand

Formula: C15H24N0O2

Molecular weight: 236.4

ThOD: 2.71 mg O2/mg

Table 1: Biological oxygen demand (BOD, mg O2/L, adjusted to nominal concentrations:

     Day 5  Day 7  Day 14  Day 21  Day 28  Day 35  Day 42
 O2 uptake of sludge (inoculum blank)  Flask 3 (B1)  14.9  17.0  21.3  25.6  25.6  25.6  25.6
   Flask 4 (B2)  16.9  19.0  23.3  25.4  25.4  25.4  27.5
   mean (B)  15.9  18.0  22.3  25.5  25.5  25.5  26.6
 O2 uptake of test (subst. + sludge)  Flask 15 (C1) 12.5   14.4  16.5  20.6  24.7  26.8  26.8
   Flask 16 (C2)  12.3  14.4  16.5  22.6  22.6  26.7  26.7
 O2 uptake of test Substance  C1 -B  -3.5  -3.6  -5.8  -4.9  -0.8  1.3  0.2
   C2 -B  -3.6  -3.6  -5.8  -2.9  -2.9  1.2  0.1
 % biodegradation of test substance  D1  -4  -4  -7  -6  -1  2  0
   D2  -4  -4  -7  -4  -4  1  0
   Mean (D)  -4  -4  -7  -5  -2  2  0

B = (B1 + B2)/2

D1 = 100 * (C1 - B)/ThOD * [S]

D2 = 100 * (C2 - B)/ThOD * [S]

D = (D1 + D2)/2

[S] = initial test substance concentration (mg/L)

Biodegradability - Respirometric method - Sodium benzoate

Theoretic al oxygen demand

Formula: C7H5O2Na1

Molecular weight: 144.1 g/mol

ThOD: 1.67 mg O2/mg

Table 2: Biological Oxygen Demand (BOD, mg O2/L, adjusted to nominal concentrations):

 

Days

5

7

14

21

28

42

BOD Sludge

Flask 3

B1

14.9

17.0

21.3

25.6

25.6

25.6

Flask 4

B2

16.9

19.0

23.3

25.4

25.4

27.5

Mean

B

15.9

18.0

22.3

25.5

25.5

26.6

BOD Refer. Subst.

Flask 1

A1

124.0

137.0

152.0

162.0

167.0

169.0

Flask 2

A2

129.0

141.0

159.0

167.0

171.0

174.0

1stfl. Corr.

A1-B

108.1

119.0

129.7

136.5

141.5

142.5

2ndfl. Corr.

A2-B

113.1

123.0

136.7

141.5

145.5

147.5

% Degr.

D1

65

71

78

82

85

86

D2

68

74

82

85

87

89

Mean

D

66

73

80

83

86

87

B = (B1 + B2)/2

D1 = 100 * (A1 - B_/ThOD * [S]

D2 = 100 * (A2 - B)/ThOD * [S]

D = (D1 + D2) / 2

[S] = Initial reference substance concentration (mg/L).

Validity criteria fulfilled:
yes
Interpretation of results:
other: No biodegradation based on oxygen consumption, but specific analysis confirms degradation of the formate ester to the corresponding alcohol product.
Conclusions:
Oxyoctaline formate undergoes, based on the oxygen consumption observed, no biodegradation after 28 days in the test conditions. Thus, Oxyoctaline formate should be regarded as not readily biodegradable according to this test.
At the test concentration Oxyoctaline Formate did inhibit the intrinsic respiration of the inoculum by more than 20% on several days of the test. Slight toxicity towards the inoculum at test concentration can therefore not be excluded.
In a non-GLP part of the study the content of the test substance flask was analysed using a specific analysis (GC-FID and GC-MS) at the end of the test (day 42). In the two replicate flasks 44.5% and 42.4% of the initial nominal concentration of Oxyoctaline Formate were found, respectively. A major degradation product was observed in both flasks which was tentatively identified as 1,3,6,7-tetramethylbicyclo(4.4.0)dec-7-ene-2-ol (= Oxyoctaline alcohol) using GC-MS analysis and comparison with library spectra. Using the relative peak areas on GC-FID chromatograms relative quantities of this degradation product were estimated to be 24.1% and 31.4% of the initial nominal concentration of oxyoctaline formate, in the two replicate flask at test end respectively.
The results show that Oxyoctaline Formate undergoes degradation to the corresponding alcohol. Based on the parent remaining, the % of primary degradation is estimated to be approximately 55% after 42 days in the test conditions.
Executive summary:

The ready biodegradability of Oxyoctaline formate was determined by Manometric Respirometry test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. Oxyoctaline formate undergoes, based on the oxygen consumption observed, no biodegradation after 28 days in the test conditions.  Thus, Oxyoctaline formate should be regarded as not readily biodegradable according to this test.

At the test concentration Oxyoctaline Formate did inhibit the intrinsic respiration of the inoculum by more than 20% on several days of the test. Slight toxicity towards the inoculum at test concentration can therefore not be excluded.

Oxyoctaline formate should be regarded as not readily biodegradatble according to this test.

In a non-GLP part of the study the content of the test substance flask was analysed using a specific analysis (GC-FID and GC-MS) at the end of the test (day 42). In the two replicate flasks 44.5% and 42.4% of the initial nominal concentration of Oxyoctaline Formate were found, respectively.  A major degradation product was observed in both flasks which was tentatively identified as 1,3,6,7-tetramethylbicyclo(4.4.0)dec-7-ene-2-ol (= Oxyoctaline alcohol) using GC-MS analysis and comparison with library spectra. Using the relative peak areas on GC-FID chromatograms relative quantities of this degradation product were estimated to be 24.1% and 31.4% of the initial nominal concentration of oxyoctaline formate, in the two replicate flask at test end respectively.

The results indicate Oxyoctaline Formate undergoes primary degradation. Based on the parent remaining, the % of primary degradation is estimated to be approximately 55% after 42 days in the test conditions.

Description of key information

The ready biodegradability of Oxyoctaline formate has been investigated in two OECD 301F ready biodegradability tests (Rudio 2003, Kreutzer 2016). Based on oxygen consumption, no biodegradation after 28 days was observed. Thus, the test substance should be regarded as not readily biodegradable according to these tests for the purposes of classification and labelling. The conclusion for the exposure assessment is that no biodegradation was observed.

Substance specific analysis was performed in the Kreutzer 2016 ready test. Primary degradation of Oxyoctaline formate to the corresponding alcohol was observed. This is taken into consideration for the purposes of the PBT assessment.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradability of Oxyoctaline formate has been investigated in two OECD301F ready screening tests (Rudio 2003, Study No. 02-E069; Kreutzer 2016, Study No. 15-E171). The test substance concentrations employed were 100 and 30 mg/L respectively. Mineralisation, determined as the amount of oxygen consumed, was not observed in either test after 28 days. Thus, the substance cannot be considered as readily biodegradable according to these two tests.

In the first study (Rudio, 2003) the toxicity was assessed, as suggested in the OECD 301F method, by comparing the respiration of a flask containing the test chemical and a readily biodegradable reference substance with the respiration of a flask containing only the readily biodegradable reference substance. Since the flask containing test substance + reference item did not have a lower respiration than the flask containing only the reference item, it was concluded that the test chemical was not inhibitory to the micro-organisms at the test concentration (100 mg/l). This concentration can be considered as a NOEC for the toxicity to STP organisms.

Specific chemical analysis was performed in the second study (Study No. 15-E171) to assess primary degradation of Oxyoctaline formate. The test was extended beyond the standard 28 days to 42 days. At the end of the test the contents of the two replicate flasks were extracted with MTBE, containing n-dodecane as an internal standard. Quantification of the residual Oxyoctaline formate using GC analysis showed 44.5% and 42.4% of the initial nominal concentration. One major degradation product was observed in the chromatograms of both flasks. This was tentatively identified as 1,3,6,7-tetramethylbicyclo(4.4.0)dec-7-ene-2-ol (2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalene-1-ol; oxyoctaline alcohol) using GC-MS analysis and comparison with library spectra. The EAWAG-BBD Pathway Prediction System supports these findings. It predicts that the most likely (aerobic) degradation pathway is to start with conversion of the ester to the corresponding alcohol with loss of formic acid / formate. Using the relative peak areas on GC-FID chromatograms (not corrected for GC-FID response factors and molecular weight), the quantities of the degradation product were estimated to be 24.1% and 31.4% of the initial nominal concentration of Oxyoctaline formate, in the two replicate flasks at test end respectively.

Based on the parent remaining, the % of primary degradation is estimated to be approximately 55% after 42 days in a ready test performed at 30 mg/L. Given that the water solubility of the registered substance is only 2.2 mg/L, the rate of biodegradation in this test will be controlled by the dissolution rate. Another important consideration is toxicity to the inoculum. At the test concentration of 30 mg/L, Oxyoctaline formate inhibited the intrinsic respiration of the inoculum by more than 20% on several days of the test. For example, on day 14 the O2 uptake in the test flask was only 16.5 mg O2/l compared to 22.3 mg O2/l in the inoculum blank. The inhibition effect is also clearly seen in the biodegradation time plots, where negative biodegradation levels (based on biological oxygen demand) are observed on most days. Slight toxicity towards the inoculum at test concentration can therefore not be excluded.

In contrast, the toxicity control performed in the previous test (Study No. 02-E069) evidenced no toxicity to the inoculum at a higher concentration (100 mg/l). However, a toxicity test which is based on the effect of a test substance on the degradation of a readily biodegradable reference item may be less sensitive than assessing the intrinsic respiration of the inoculum to the test substance alone. Two key issues in using the former method to evaluate the toxicity of a substance to be assessed for biodegradability are described by Reynolds et al (1987). The first is that for easily degradable substrates the initial number of active microorganisms in the inoculum will not be critical. Even if a large proportion of the inoculum is killed, the remaining organisms will proliferate and degrade the substance, still giving a high degradation rate with no indication of the toxic effect of the test substance added. The second is that the effect of the test substance on the degradation of a reference substance may not reflect the effects of the test substance on those microorganism species responsible for degrading it. Furthermore, the biodegradation time plot in Study No. 02-E069 also showed negative degradation levels of -1 to -3% throughout the test indicating some inhibition of the intrinsic respiration. Therefore, the registrant considers that the influence of toxicity on the results of the two ready screening tests cannot be ruled out for this substance.  

Biodegradation screening tests are performed under very stringent conditions compared to relevant environmental conditions. Thus the results from such tests are considered conservative and worst case.  Therefore, it is expected that under realistic environmental exposure, more efficient and rapid biodegradation kinetics will be observed.

 

Reynolds L., Blok J., de Morsier A., Gerike P., Wellens H., Bontinck, W.J. (1987). Evaluation of the toxicity of substances to be assessed for biodegradability. Chemosphere, 16, 2259.