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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 20 AUG 2007 to 3 DEC 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
No analytical monitoring
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Based on the information provided by the Sponsor, the test item is soluble in water at a concentration >1 g/L. Therefore, 502.17 mg of the test item was dissolved in 284 mL tap water by ultrasonic treatment for five minutes and intense stirring for 15 minutes at room temperature. In this way a clear solution was obtained. Then, 16 mL synthetic wastewater and 200 mL activated sludge inoculum were added.
- Differential loadings: No (limit test)
- Controls: two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the single test concentration of the test item under identical test conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: no, aerobic activated sludge came from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, CH) treating predominantly domestic wastewater.
- Method of cultivation: During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater* per liter and was kept at room temperature under continuous aeration until use. The pH of the activated sludge inoculum was 6.5.
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter.
- Pretreatment: no
- Initial biomass concentration: 3 g dry material per liter

* Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 × 2H2O
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
filled up to a final volume of 1 liter with deionized water

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
none
Hardness:
No data
Test temperature:
The temperature measured in test media in one control was 20°C at the start and at the end of the incubation period
pH:
7.3 - 7.9 (test item)
7.2 - 8.1 (reference item)

Dissolved oxygen:
8.1 - 8.8 mg/L (test item)
8.5 - 8.9 mg/L (reference item)


Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentration: 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: The test was performed in 2000-mL glass beakers, filled with a 500-ml mixture containing synthetic wastewater, test medium and activated sludge.
- Aeration: During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated by intense stirring on magnetic stirrers, to avoid possible foaming and/or stripping of the test item.
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: sludge concentration = The inoculum had a sludge concentration of 2.2 g/L dry weight (corresponding to about 0.9 g dry material per liter test medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement:The pH values and dissolved oxygen concentrations were determined after the addition of synthetic wastewater and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic wastewater, the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Lighting: no data

EFFECT PARAMETERS MEASURED : For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: a limit test has been performed to demonstrate that the test item has no toxic effect on activated sludge up to the nominal concentration of 1000 mg/L.
- Range finding study: none
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol tested at 5, 16 and 50 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The test item Dimethyl 2-Methyl Glutarate had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 1000 mg/L.
Thus, the 3-hour NOEC (EC15) of Dimethyl 2-Methyl Glutarate to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 14 mg/L (the 95% confidence limits could not be calculated). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
The 3h-EC50, -EC20 and -EC80 of the test item and their 95% confidence limits could not be calculated due to the absence of a toxic effect. For the reference substance, the probit analysis was used.

Table 1 : Influence of Dimethyl 2 -methyl glutarate and 3, 5-dichlorophenol on the oxygen consumption of activated sludge

 

Vessel

No.

Test chemical

Nominal concentration of test chemical

(mg/L)

Oxygen consumption rate

(mg O2/L min-1)

Inhibition

(%)

A

B

Control

Control

0

0

1.033

1.062

 

Mean

Deviation (%)

 

 

1.047

2.8

 

1

2

3

5

3,5-dichlorophenol

3,5-dichlorophenol

3,5-dichlorophenol

Dimethyl 2 -methyl glutarate

5

16

50

1000

0.937

0.354

0.105

0.975

10.6

66.2

90.0

6.9

 

-% inhibition: increased oxygen consumption rate relative to control

Validity criteria fulfilled:
yes
Remarks:
The EC50 of the reference substance is in the acceptance range and the oxygen consumption rates of the 2 controls differed by less than 15% (3%).
Conclusions:
Dimethyl 2-methyl glutarate has no toxic effect on the respiration rate of activated sludge microorganisms at the limit test concentration of 1000 mg/L.
Executive summary:

The inhibitory effect of DIMETHYL 2-METHYL GLUTARATE on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209.

A limit test was performed with one concentration of DIMETHYL 2-METHYL GLUTARATE of nominal 1000 mg/L.

In addition, two controls and three different concentrations of the reference item 3,5 dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. After a 3 -hour incubation time, the inhibition of the respiration rates were investigated via a determination of oxygen consumption rates.

The results of the reference treatments (EC50 = 14 mg/L within the guideline-recommended range of 5 - 30 mg/L) confirmed suitability of the activated sludge and the method used.

The test item DIMETHYL 2-METHYL GLUTARATE had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Thus, the 3-hour NOEC of DIMETHYL 2-METHYL GLUTARATE to activated sludge microorganisms was > = 1000 mg/L. The 3-hour EC20, EC50 and EC80 were clearly higher than 1000 mg/L.

Description of key information

The 3-hour NOEC of dimethyl 2-methyl glutarate to activated sludge microorganisms was > = 1000 mg/L. The 3-hour EC20, EC50 and EC80 were clearly higher than 1000 mg/L. Hence, dimethyl 2-methyl glutarate has no toxic effect on the respiration rate of activated sludge microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

One experimental study, scored as Klimisch 1, is available (Seyfried B., 2007) and selected as a key study.