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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: yes, Municipal activated sludge taken from a laboratory wastewater treatment plant fed with municipal sewage.
- Preparation of inoculum for exposure: The inoculum was washed, brought to a concentration of 5 g/L dry substance and aerated during the night. 50 ml were added to a total volume of 250 ml to obtain a concentration of 1 g/L dry substance in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
180 min
Test temperature:
20 ± 2 °C
Dissolved oxygen:
Oxygen concentration during aeration: > 2,5 mg/l
Oxygen concentration immediately before measurement: > 6,5 mg/l
Nominal and measured concentrations:
0, 62.5, 125, 250, 500, 1000 mg/l nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer-vessel (nominal volume 250 ml)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Synthetic medium: 8 ml/vessel 100-fold concentrated OECD medium
Reference substance (positive control):
yes
Remarks:
dichlorophenol
Duration:
180 min
Dose descriptor:
EC10
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:
180 min
Dose descriptor:
EC10
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Al
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
180 min
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
Duration:
180 min
Dose descriptor:
EC50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Al
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
satisfactory
Validity criteria fulfilled:
yes
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A clear guideline was followed, however little data on the substance identity and test organisms was presented so it is not 100% sure that the Al2(SO4)3, was the actual measured toxicant. Also no control group was used so effects, although a slim chance) might also be due to other conditions.
Qualifier:
according to guideline
Guideline:
other: Romanenko, V. I., Velichko, I. A.: Effect of chromium ions on vital activity of bacteria and algae. In: The biology of inland waters, No. 21 (in Russian). Nauka, Leningrad, 1974: 12. 8.
Qualifier:
according to guideline
Guideline:
other: Romanenko, V. I., Zhonson , T. D., Mikryakov, V. R.: Methods of utilizing heterotrophic assimilation of CO2 in determining the effect of pesticides on vital activity of bacteria. In: The biology of inland waters, 44. (In Russian). Nauka, Leningrad, 197
Qualifier:
according to guideline
Guideline:
other: Panasenkov, Yu. V.: Determination of the effect of sewage waters of a cellulose plant on natural microbial water coenoses according to absorption of 14-CO2 In: The biology of inland waters, 61. (In Russian) Nauka, Leningrad, 1983: 7.
Principles of method if other than guideline:
exposure time is 24h
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
After exposure and fixing with Lugol's solution, 5 filters
were obtained from each bottle. To remove the labelled carbonate adsorbed on the surface of
the bacterial cells and the ultrafilters, they were washed in 3 % HCI solution. Then the filters
were dried. After that, no sorption of labelled carbonate was observed on the filter and the
surface of the cells.
Vehicle:
not specified
Details on test solutions:
The water taken from the reservoir is
distributed into 20-litre tanks into which aluminium sulphate is added in concentrations of
0.1, 1, 10 and 100 mI/L, respectively.
Test organisms (species):
aerobic microorganisms
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
24 h
Nominal and measured concentrations:
nominal: 0.1, 1.0, 10, 100 mg/L
Details on test conditions:
The experiment is arranged in two parallels, i.e.,
there are two tanks of each concentration. After a fixed period (the experiment takes 24
hours), water samples are collected from the tanks for inoculation on nutrient media to
assess the amount off saprophytes (standard method) and to determine heterotrophic
assimilation and intensity of photosynthesis. The assessment of the amount of saprophytic
microflora is carried out on 10 Petri dishes for each concentration.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Reported statistics and error estimates:
According to these values and using V. 1. Romanenko's correlations
it was possible to calculate bacterial production. Statistical analysis of the obtained data
consisted in the calculation of the means and the value of the P > 0.95 confidence interval
using the methods of biological statistics. Statistical analyses of the values of radioactivity
of the bacteria have shown that the values of confidence intervals are negligible.
This justified the omission of a more detailed analysis of the obtained results.

Results of our studies demonstrate that the concentrations of 0.1 and 1 mg/L have no effect on the microorganisms while the concentration of 10 mg/L reduces the number of saprophytes by 60 %. It can be seen that even a concentration

of 1 mg/L of aluminium sulphate has some influence on the fixation of labelled carbonic acid by microorganisms. Further increase in the concentration of the toxicant leads to a decrease in CO2 absorption. On the basis of results

of the experiment, it can be stated that the concentration of aluminium sulphate which has no effect on natural microcoenosis (inactive concentration) amounts to 1 mg/L. The threshold concentration of the toxicant can be found within the interval between 1 and 10 mg/L.

At the mentioned concentration, heterotrophic fixation of 14-CO2 by microorganisms

decreased by 50 % and the number of saprophytes decreased by 60 %.

This confirms once again the possibility of using the radiocarbon method of estimating

bacterial production in water toxicology. The effect of the concentration of 100 mg/L on the microbial coenosis is

so much greater that it causes its destruction.

Validity criteria fulfilled:
not applicable
Conclusions:
The EC50 was determined to be 8 mg/L, the LOEC was determined to be 1 mg/L nominal. The usebalitlity of the data for risk assessment purposes is questionable due to the missing substance indentity data and the fact that no control group was used. It is therefore adviced to use this data only as supporting and not as keystudy.
Executive summary:

The methods of determining the effect of chemical compounds on the processes of

microbial self-cleaning of waters are mainly based on the calculation of the number of indicator

microorganisms grown after being incubated on complex nutrient media following exposure of the

waters with the substances under study. These methods examine the reaction of specific

microorganisms under conditions rather different from those really existing in the water

reservoir. The author proposes to use the method of heterotrophic assimilation of CO2 for the

determination of the activity of natural bacterial associations in the study of the effect on

them of both multicomponent sewage waters of industrial plants and specific chemical compounds.

The determination of the values of bacterial productivity according to heterotrophic assimilation

of CO2 by natural microbial coenosis was used as the criterion of the effect of the toxicant..

Toxicity of the substance under study is evaluated according to its inhibitory effect on the

production of natural bacteriocoenosis (test object). Field of application: water toxicology,

supervision of toxicity of aqueous media, evaluation of the quality of water.

Standard experiment examining the effect of aluminium sulphate on natural bacterial

coenosis is considered an example. It has been demonstrated that the effect of aluminium

sulphate begins to be felt in concentrations starting from 1 mg/L. Statistical analysis

of results was carried out by calculating the mean value and confidence intervals (EC50 was determined to be 8 mg/L, nominal).

The investigations have shown that application of the method of heterotrophic assimilation

of CO2 for these purposes is feasible and of considerable interest with a view to its high

sensitivity and precision.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
25 February 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to an appropriate OECD test guideline, and in compliance with GLP. The study is considered to be reliability 1 (reliable without restrictions); the read across of the result is considered to be reliability 2 (reliable with restrictions).
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of 202028/A tested was a clear colourless liquid and was treated as 100% pure.
A stock solution of 0.5 g/l was prepared by adding 125.4 mg test substance to 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (15 minutes) was used to accelerate dissolution and to ensure homogeneity. Volumes of the clear colourless stock solution, without flocks, corresponding to the test concentration were then added to the test media. A concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (4.0 g/l of sludge, as used for the test). The pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours exposure period.
Test temperature:
between 18.5 and 19.1°C
pH:
between 7.8 and 8.0
Dissolved oxygen:
Oxygen consumption:
Control series:66-68 mg O2/l/h
Test substance series: 63-66 mg O2/l/h
The oxygen concentration at the start of measurements was at least 7.2 mg O2/l
Nominal and measured concentrations:
Nominal concentration: 100 mg/l
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 ml oxygen bottles (measuring) and
1 litre test bottles(exposure)
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2/ series
- Biomass loading rate:1.6 g/l suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO water)
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/l.

TEST CONCENTRATIONS
- Test concentrations: limit study at 100 mg/l
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol)
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
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 4.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Al
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 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:
EC10
Effect conc.:
> 4.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Al
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
No significant inhibition of respiration rate of the sludge was recorded at 100 mg 202028/A per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of 202028/A exceeded 100 mg/l (based on nominal concentrations).
Results with reference substance (positive control):
Results with reference substance valid: Yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing concentration , ranging from a 29 % inhibition at 5.0 mg/l to 84 % at 30 mg/l. Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 9.0 mg/l
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, 202028/A was not toxic to waste water (activated sludge) bacteria at 100 mg/l. The 3-hour EC50 of 202028/A exceeded 100 mg/l (based on nominal concentrations).
Based on aluminum element the EC50 and EC10 are greater than 4.4 mg/l (total Al)

Description of key information

Key value for chemical safety assessment

Additional information

One activated sludge respiration inhibition test according to OECD 209 is available with aluminum chloride according to standard guideline (Schwarz, 2008). No effects were seen up to the highest test concentration of 1000 mg/l test material. The test substance contained 20 g Al/100 g and the test concentrations can be converted to mg/l Al. 1000 mg/l test material is equal to 200 mg/l Al. A second activated sludge respiration inhibition test is available for aluminum chloride hydroxide sulfate according to standard guideline (Desmares-Koopmans, 2010). It was performed as limit test at 100 mg/l test material. The test substance contained 4.4% Al and the test concentrations can be converted to mg/l Al. 100 mg/l test material is equal to 4.4 mg/l Al. Both tests available did not show any effect to microorganisms.