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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Florhydral
- Analytical purity: 97.6% (GC)
- Lot/batch No.: 213739
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Aire).

The sludge was collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and re-suspending in mineral medium) and maintained aerobic until being used on the same day.

The dry weight of suspended solids was 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.

Dry weight of suspended solids : 0.745 g/L.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
A volume of suspended sludge corresponding to 25 mg dry weight is placed in a 250 mL volumetric flask and made-up with mineral medium. This is poured in to each test flask of the SAPROMAT. The test and reference substance are weighed in small aluminium boats which are added to the test flask contents. The pH of each flask is measured, and, if necessary, adjusted to 7.4 +/- 0.2 with Phosphoric Acid or Potassium Hydroxide. 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. Following temperature and pressure equilibration, the oxygen meters of the instrument are set to zero (time zero of the experiment).
Reference substance:
aniline
Remarks:
Aniline - Merck, Darmstadt, Art. No. 1261; Purity : Minimum 99.5% Test Concentration : 100 mg/L
Parameter:
% degradation (O2 consumption)
Value:
18
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
28
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
76
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
104
Sampling time:
28 d
Results with reference substance:
Reference Substance (Aniline) fulfilled > 40% biodegradation on Day 7 and > 65% by Day 14.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
FLORHYDRAL was found to be INHERENTLY biodegradable (104% biodegradation after 28 Days).
Executive summary:

The Inherent Biodegradability of FLORHYDRAL has been determined by the Respirometric Method (Modified MITI Test II) according to the OECD Guidelines for Testing of Chemicals, Method No. 302C.


The biodegradation rate of FLORHYDRAL after 28 days is 100% in the test conditions.  Therefore, FLORHYDRAL can be regarded as INHERENTLY biodegradable.


Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
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. SC00002800;
Purity 98.3%;
Expiry Date : 23 May, 2012
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, 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 was 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.

The dry weight of suspended solids was 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.

Dry weight of suspended solids: 4.05 g/L, diluted to 1.53 g/L.
Duration of test (contact time):
62 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test substance samples (7.65 mg, corresponding to 30.0 mg/l in 255 ml of test medium) were weighed in small aluminium boats and added directly to the test flasks of the Oxitop, whereas the reference substance (sodium benzoate) was added as 1.00 ml of a 10.2 g/l solution in mineral medium, to give a total volume of 103 ml.

Flasks were filled with 250 ml of mineral medium (flasks containing reference substance: 100 ml). Samples of test or reference substance are added. Then suspended sludge diluted to a concentration of 1.53 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 test flasks, and the test flasks are placed in the temperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

Everyday the oxygen consumption of each flask was recorded and correct temperature and stirring are checked.

At the end of the test period (normally 28 days), the pH of each flask was measured again.
Reference substance:
benzoic acid, sodium salt
Remarks:
Reference substance tested at 100 mg ref substance/L concentration.
Parameter:
% degradation (O2 consumption)
Value:
16
Sampling time:
14 d
Remarks on result:
other: Start of 10-d window
Parameter:
% degradation (O2 consumption)
Value:
68
Sampling time:
24 d
Remarks on result:
other: End of 10-d window
Key result
Parameter:
% degradation (O2 consumption)
Value:
74
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
79
Sampling time:
62 d
Details on results:
Florhydral undergoes 74 % biodegradation after 28 days (79% after 62 days) in the test conditions. The 10-day window criterion is also fulfilled (16% biodegradation on day 14 and 68% on day 24).

Thus, Florhydral should be regarded as readily biodegradable according to this test.

Florhydral did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.
Results with reference substance:
Reference Substance (Sodium Benzoate) attained 85% biodegradation by Day 7 and 93% by Day 14.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
FLORHYDRAL is READILY Biodegradable (74% biodegradation on Day 28, fulfilling the 10-Day window criteria).
Executive summary:


Florhydral undergoes 74 % biodegradation after 28 days (79% after 62 days) in the test conditions. The 10-day window criterion is also fulfilled (16% biodegradation on day 14 and 68% on day 24).


 


Thus, Florhydral should be regarded as readily biodegradable according to this test.


 

Florhydral did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08 February, 2012 to 30 March, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
FLORHYDRAL :
Lot No. :SC00003466;
Purity : 99.2% (GC area %);
Date of Expiry : 22 August, 2012
Oxygen conditions:
aerobic
Inoculum or test system:
other: Collection of incoulum (sewage sludge, water, sediment) - described in detail, below.
Details on inoculum:
Activated sludge :
On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes, and inland sea; or return sludges from sewage plants). Activated sludge, which was prepared and controlled in this laboratory ), and was used in this study [sampling period: December, 2011, initiation date of use: January 10, 2012 ]. The activated sludge, which was cultivated for 23 hours after the synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0 +/- 1.0.

Measurement of concentration of suspended solid in activated sludge and decision of additive amount of activated sludge :
Additive amount of activated sludge into the test vessel was 2.62 mL on the basis of the concentration of suspended solid in the activated sludge which was determined by the following methods;
Method In accordance with Japanese Industrial Standards (JIS) K 0102-2010
Date February 6, 2012
Result 3430 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
Substance specific analytics for Parent and corresponding primary metabolites.
Details on study design:
Preparation of test solutions :
The following test solutions were prepared and cultured :
a) Addition of test item or aniline
1) Test solution (water + test item) (n=1, Vessel No. 1)
In one test vessel, 30 mg of the test sample was accurately weighed and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L
2) Test solution (sludge + test item) (n=3, Vessel Nos. 2, 3 and 4)
In each test vessel, 30 mg of the test sample was accurately weighed and added to the basal culture medium [the volume subtracting the volume (2.62 mL) of activated sludge from 300 mL], so that the concentration of the test item reached 100 mg/L.
3) Test solution (sludge + aniline) (n=1, Vessel No. 6)
In one test vessel, 29.5 µL (30 mg) of aniline was removed by using a microsyringe and added to the basal culture medium [the volume subtracting the volume (2.62 mL) of activated sludge from 300 mL], so that the concentration of aniline reached 100 mg/L.
4) Test solution (control blank) (n=1, Vessel No. 5)
In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (2.62 mL) of activated sludge from 300 mL].
b) Inoculation of activated sludge
The activated sludge was added to each test vessel described in 2), 3), and 4), so that the concentration of the suspended solid reached 30 mg/L.


Conditions of cultivation
Cultivation temperature 25 +/-1ºC
Cultivation duration 28 days (under dark conditions)
Stirring method Each test solution was stirred by a stirrer.
Reference substance:
aniline
Remarks:
Aniline : Lot No. : DCP5097 Purity : 99.5% Density : 1.022 g/mL (at 20°C)
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
98
Sampling time:
28 d
Remarks on result:
other: Parent : 2-3% of the applied dose at Time 0 remained as Parent following 28 days of incubation.
Remarks:
A Mass Balance of 94 - 97% of the originally applied dose was obtained on Day 28, constituting : Parent Remaining : 2 - 3% GR-81-6079 (Carboxylic Acid Metabolite) : 72 - 77% GR-82-4337 (Alcohol Metabolite) : 18 - 22%
Details on results:
A Mass Balance of 94 - 97% of the originally applied dose was obtained on Day 28, constituting :
Parent Remaining : 2 - 3%
GR-81-6079 (Carboxylic Acid Metabolite) : 72 - 77%
GR-82-4337 (Alcohol Metabolite) : 18 - 22%
Results with reference substance:
Percentage Biodegradation of Aniline :
Day 7 = 53%
Day 14 = 71%

Percentage biodegradation after 28 days was as follows.

The percentage decrease of the test item, the ratio of decreased amount to the theoretical amount, was calculated instead of the percentage biodegradation by the chemical analysis because the percentage residue of the test item in the test solution (water + test item) (73%) was less than 90%.



*4 The average percentage biodegradation was regarded as 0% because the calculated average value shown in parentheses was negative.


Analytical results of the test solutions and the CO2 absorbents after 28 days were as follows :


*3       The value of the test solution (control blank) was subtracted from the values of the test solutions (sludge + test item).



Discussion


Results of analysis for test item and converted products in test solution


The percentage residue of the test item was 73% in the test solution (water + test item), and 2-3% in the test solutions (sludge + test item).  In addition, the average percentage biodegradation by BOD in this study was 0%. From these results, it was considered that converted products were produced in the test solutions. Therefore, GR-81-6079 and GR-82-4337, which are expected to be produced by oxidation or reduction of the test item, were determined. The percentage production of GR-81-6079 was 4% in the test solution (water + test item), and 72-77% in the test solutions (sludge + test item). The percentage production of GR-82-4337 was 0% in the test solution (water + test item), and 18-20% in the test solutions (sludge + test item).


The mass balance, which was the sum of the percentage productions of the converted products and the percentage residue of the test item, was 94-97% in the test solutions (sludge + test item). But the mass balance was 77% in the test solution (water + test item).


Influence of CO2 absorbent


The mass balance was lost in the test solution (water + test item). No peak except for the test item and GR-81-6079 was detected on the chromatogram of HPLC analysis for the test solution (water + test item). From these results, it was presumed that some of the test item and the converted products were adsorbed to the CO2 absorbent, which was installed in the test solution for the measurement of BOD. The qualitative analysis of the test item and the converted products in the CO2 absorbent was performed, but they were not detected. 


The mass balance, which was the sum of the percentage production of GR-81-6079 and the percentage residue of the test item, was 102% in the test solution (water + test item) without the CO2 absorbent (additional test). Therefore, the loss of the mass balance in the test solution (water + test item) might be caused by the strong adsorption of the test item and the converted product to the CO2 absorbent, and the interference of the extraction of them from the CO2 absorbent.

It is suggested that the test item, GR-81-6079 and GR-82-4337 were retained in the test solution (sludge + test item) because the mass balance was 94-97%. Therefore, it is judged that the CO2 absorbent did not affect the evaluation of biodegradability.



Summary

Under the test conditions of this study, almost all the test item underwent primary degradation and was converted to 3-(3-isopropylphenyl)butanoic acid and 3-(3-isopropylphenyl)butanol. The converted products and the rest of the test item did not undergo complete degradation under the test conditions and during the 28 Day incubation period. No conclusion can be drawn on the degradation rate kinetics of the primary degradation products of Florhydral given that substance specific analyses are performed at one single, punctual timepoint at the end of the incubation period on Day 28. Because oxygen consumption in the test solution (Control Blank) was higher than in the test solution (sludge + test item) triplicate samples, it appears that the test item and/or the converted products may have exerted inhibition effects on the microbial population of the sludge inoculum at the test concentration level tested.


Conversion of test item in test solutions (sludge + test item) (presumption) at Day 28 of Incubation :





Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions of this study, almost all the test item underwent primary degradation and was converted to 3-(3-isopropylphenyl)butanoic acid and 3-(3-isopropylphenyl)butanol. The converted products and the rest of the test item did not undergo complete degradation under the test conditions and during the 28 Day incubation period. No conclusion can be drawn on the degradation rate kinetics of the primary degradation products of Florhydral given that substance specific analyses are performed at one single, punctual timepoint at the end of the incubation period on Day 28.

Description of key information

BIODEGRADATION :

FLORHYDRAL was found to be READILY BIODEGRADABLE (74% biodegradation after 28 Days, fulfilling the 10-Day window criteria) under the strict testing conditions of the OECD 301F guideline, and was also observed in a separate OECD 302C study to undergo 100% biodegradation during the 28 day test period.


FLORHYDRAL is a primary aldehyde and under typical aerobic environmental conditions will be rapidly oxidised to the Carboxylic acid metabolite (GR-81-6079), which then undergoes mineralisation (c.f. results of the OECD 301F Ready Biodegradation study)

.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

BIODEGRADATION :

FLORHYDRAL was found to be READILY BIODEGRADABLE (74% biodegradation after 28 Days, fulfilling the 10-Day window criteria) under the strict testing conditions of the OECD 301F guideline, and was also observed in a separate OECD 302C study to undergo 100% biodegradation during the 28 day test period.


FLORHYDRAL is a primary aldehyde and under typical aerobic environmental conditions will be rapidly oxidised to the Carboxylic acid metabolite (GR-81-6079), which then undergoes mineralisation (c.f. results of the OECD 301F Ready Biodegradation study)

.