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Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-11-01 - 2005-02-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test had been conducted according to relevant guidelines and compliant to GLP. The results are plausible and well documented.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
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)
Remarks:
Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5] based on OECD-GLP 1997 (C(97) 186/Final)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
not applicable

Test solutions

Vehicle:
no
Details on test solutions:
A limit test was performed in accordance with the EU Commission Directive 92/69/EEC (C.1, C.2, and C.3) and the OECD Series on Testing and Assessment No. 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000) to demonstrate that the test item has no toxic effect on activated sludge up to the nominal concentration of 100 mg/L. Thus, only one test item concentration of 100 mg/L was tested.
Due to the limited water solubility of the test item, no stock solution was prepared. An amount of 49.98 mg of the test item was weighed (by means of an analytical balance), transferred to the test flask with 284 mL of tap water, and mixed into the tap water by ultrasonic treatment for ten minutes and intense stirring for 24 hours at room temperature in the dank, to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After the stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added.
Additionally, two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the limit test concentration of the test item, under otherwise identical test conditions.
The reference item 3,5-dichlorophenol was tested in parallel under otherwise identical test conditions, and functioned as a positive control. The nominal concentrations tested were 5, 16 and 50 mg/L. A stock solution of 3,5-dichlorophenol was prepared according to the test guidelines. The pH was determined to be 7.7. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and tap water in the respective test flasks to obtain the desired concentrations.

EU Commission Directive 92/69/EEC, C.1 - C.3, 1992
OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and mixtures

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. To eliminate possible inhibitory material, the sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based an this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of three 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. Before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was 7.0.

* Synthetic wastewater:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCI
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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
not applicable
Test temperature:
20°C
pH:
Controls: 7.2-8.2
3,5-dichlorophenol: 7.4-8.3
Treatment: 7.3 (start) - 8.1 (end)
Dissolved oxygen:
[mg/L]
Controls: 7.8-8.4
3,5-dichlorophenol: 7.7-8.8
Treatment: 8.5 (start) - 7.6 (end)
Salinity:
not applicable
Nominal and measured concentrations:
Due to the poor solubility of the test item a limit test at nominal 100 mg/L had been performed. Analytical verification of the acutal concentration was not possible.
Details on test conditions:
The test was performed in 1000-mL glass flasks. The test vessels were labeled with the necessary information to ensure unmistakable identification.
At the start of the test (after the stirring period of 24 h), synthetic wastewater and activated sludge inoculum were added. The inoculum was diluted with tap water to a sludge concentration of 4.0 g/L dry weight (corresponding to about 1.6 g dry material per liter test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test suspension of the test item, and finally to the second control.
During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated with compressed air at a flow of approximately one liter per minute. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before measurement of the respiration rates, the dissolved oxygen concentrations were at least 7.6 mg/L. The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period
For measurement of the respiration rate, a well-mixed sample of each test medium was poured into a BOD-flask alter exactly 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 for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/(L*minute)) was determined from the linear part of the respiration curve in the range of 6.5-2.5 mg 02/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.
Determination of inhibition rates follwed exactly the procedure described in the guideline.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Due to poor water solubility of the test item, a limit test at 100 mg/L nominal concentration directly added to the test flasks had been performed
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Due to poor water solubility of the test item, a limit test at 100 mg/L nominal concentration directly added to the test flasks had been performed
Details on results:
At the limit test concentration of 100 mg/L, the test item was unevenly suspended in the test medium. Thus, this test concentration was clearly above the water solubility limit of the test item under the test conditions.
At the limit test concentration of 100 mg/L, the test item had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. Based an the physical appearance of the test suspension, the saturation concentration (water solubility limit of the test item under the test conditions) was <100 mg/L, and resulted in less than 15% inhibition after three hours contact time. Thus, the 3-hour NOEC of the test item to activated sludge microorganisms was at the limit of water solubility of the test itemunder the test conditions, or at 100 mg/L. The 3-hour EC20, EC50 and EC80 were clearly higher than the limit of water solubility of the test item under the test conditions, or higher than 100 mg/L.
The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 10% (guideline-recommended maximum variation: 15%).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 12 mg/L (the 95% confidence limits were not calculable). 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:
not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
A respiration inhibition test had been performed according to relevant guidelines and compliant to GLP (reliability category 1). The test was conducted as a limit test at 100 mg/L nominal concentration added directily to the test vessels as the test item is of very poor water solubility. At the limit concentration, the test item had no significant inhibitory effect on respiration rate of activated sludge as compared to the controls (1.8% after 3 hours).
Results with the reference substance 3,5-dichlorophenol with an EC50 (3 hours) of 12 mg/L confirmed the suitability of the test.
Executive summary:

The inhibitory effect of the test item 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.

One nominal concentration of 100 mg test item per liter was tested. This limit test was performed in accordance with the EU Commission Directive 92/69/EEC and the OECD Series on Testing and Assessment No. 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000) to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 100 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. The results of these treatments confirmed suitability of the activated sludge and the method used.

At the limit test concentration of 100 mg/L, the test item had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of the test item under the test conditions) was <100 mg/L, and resulted in less than 15% inhibition after three hours contact time. Thus, the 3-hour NOEC of the test item to activated sludge microorganisms was at the limit of water solubility of the test item under the test conditions, or at 100 mg/L. The 3-hour EC20, EC50 and EC80 were clearly higher than the limit of water solubility of the test item under the test conditions, or higher than 100 mg/L.