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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The applied method is taken from a standard procedure (Owen 1976, Water Research 13) and the experimental settings are given in sufficient detail t consider the results to be reliable with restrictions.
Qualifier:
no guideline followed
Principles of method if other than guideline:
A standard anaerobic toxicity assay for methanogens was performed as described by Owen et al. (1979) in "Bioassay for monitoring biochemical methane potential and anaerobic toxicity", Water Research 13, 485.
end-point = inhibition of metabolic rate
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,2,3-Trichloropropane
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
All tests were carried out in sealed 125-mL serum bottles to prevent loss of volatile chemicals. A range of toxicant concentrations were used in each experiment to identify by interpolation the concentration that inhibited the culture by 50 % compared to the uninhibited controls.
Test organisms (species):
anaerobic bacteria
Details on inoculum:
An enrichment culture of methanogens was used that had been maintained in the laboratory for over 10 years. The 400-L, complete mix reactor was operated at a temperature of 35 °C. The solids and hydraulic retention times were both 50 days. The reactor was fed acetate (50000 mg/L) as a sole organic carbon source in a buffered inorganic nutrient solution once per day.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
35 °C
pH:
7
Nominal and measured concentrations:
All tests were carried out in sealed 125-mL serum bottles to prevent loss of volatile chemicals. A range of toxicant concentrations were used in each experiment to identify by interpolation the concentration that inhibited the culture by 50 % compared to the uninhibited controls.
Reference substance (positive control):
not required
Remarks:
A set of 127 chemicals was tested.
Duration:
48 h
Dose descriptor:
IC50
Effect conc.:
0.63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of gas production
Validity criteria fulfilled:
not applicable
Conclusions:
The gas production under anaerobic conditions of methanogens was inhibited by 50 % at a TCP concentration of 0.63 mg/L.
Executive summary:

An anaerobic toxicity assay was carried out with TCP using methanogens in a static test over a period of 48 hours at a temperature of 35 °C according to the principles laid down by Owen et al (1979). The criterion for toxicity was the inhibition of gas production. A TCP concentration of 0.63 mg/L inhibited the gas production by 50 % after 48 hours and was identified as the IC50 value.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test was not carried out according to standard procedures, but was designed to mimic the standard toxicity assays by using cell concentrations typical of standard assays such as the ETAD assay. The time period of observation was extended beyond the 3 hours of the standard respiration inhibition test to account for effects on cell growth in addition to respiration. The experimental settings and test conditions are documented in sufficient detail. The results are considered to be reliable with restrictions.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
extended observation period, higher temperature
Principles of method if other than guideline:
end-point = inhibition of metabolic rate
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,2,3-Trichloropropane
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
All test were carried out in sealed 125-mL serum bottles to prevent loss of volatile chemicals. A range of toxicant concentrations were used in each experiment to identify by interpolation the concentration that inhibited the culture by 50 % compared to the uninhibited controls.
Test organisms (species):
activated sludge
Details on inoculum:
Seed bacteria for the aerobic heterotroph culture were obtained from the mixed liquor of an activated sludge wastewater treatment plant. The reactor was a 20-L glass vessel. Diffused aeration provided complete mixing and aeration. The reactor was fed with a complex carbon source consisting of infant formula and additional inorganic salts. The COD of the feed solution was approximately 3800 mg/L. Six equal 10-minute feedings were given per day. The hydraulic retention time equalled the solids retention time of 5 days. The nitrogen concentration was restricted to that necessary for heterotrophic cell growth. The nitrifier population was estimated to be only 1 % of the total bacteria population.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
24 h
Test temperature:
25 °C and 35 °C
pH:
7
Nominal and measured concentrations:
Series of concentrations, but no data
Details on test conditions:
All tests were carried out in sealed 125-mL serum bottles. Cell concentrations between 200 and 1800 mg/L typical of the ETAD standard assay were used. A small test tube containing sodium hydroxide solution was placed in each serum bottle to absorb the carbon dioxide produced in aerobic metabolism. The bottles were surcharged with 50 mL of pure oxygen and placed on a shaker to ensure that oxygen transfer did not limit the rate of activity. The decrease in the gas volume measured at approximately 0.5-day intervals was used as the measure of activity.
Reference substance (positive control):
not required
Remarks:
A set of 73 substances was tested.
Duration:
24 h
Dose descriptor:
IC50
Effect conc.:
290 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Validity criteria fulfilled:
not applicable
Conclusions:
A concentration of 290mg/L TCP caused an inhibition of the respiration of aerobic heterotrophs by 50 % compared to uninhibited controls in the respiration inhibition test carried out with activated sludge.
Executive summary:

A modified respiratory inhibition test on TCP was carried out with activated sludge obtained from a wastewater treatment plant. The study is generally comparable to the OECD TGD 209 standards. The test was not carried out according to standard procedures, but was designed to mimic the standard toxicity assays by using cell concentrations typical of standard assays such as the ETAD assay. The time period of observation was extended beyond the 3 hours of the standard respiration inhibition test to account for effects on cell growth in addition to respiration. The experimental settings and test conditions are documented in sufficient detail. The results are considered to be reliable with restrictions.

Sealed 125-mL serum bottles supplied with 50 mL of additional pure oxygen were used in the test. The toxic effect on aerobic heterotrophs was determined by measuring the oxygen demand at approximately 0.5 -days intervals. It was found that after 24 hours the respiration of aerobic bacteria was inhibited by 50 % at a concentration of 290 mg/L, which was taken as the IC50 value.

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was carried out following principles that are documented in sufficient detail in the publication to consider the experimental results to be reliable with restrictions.
Qualifier:
equivalent or similar to guideline
Guideline:
ISO 9509 (Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms)
Principles of method if other than guideline:
The test measures the inhibition of ammonia consumption as the criterion for toxic inhibition of Nitrosomonas
end-point = inhibition of metabolic rate
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,2,3-Trichloropropane
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
All tests were carried out in sealed 125-mL serum bottles to prevent loss of volatile chemicals. A range of toxicant concentrations were used in each experiment to identify by interpolation the concentration that inhibited the culture by 50 % compared to the uninhibited controls.
Test organisms (species):
Nitrosomonas sp.
Details on inoculum:
The seed bacteria for the nitrifying enrichment culture was obtained from the mixed liquor of an activated sludge wastewater treatment plant treating meat-packing, rendering, and hide-curing wastewater. The reactor vessel was a 20-L glass bottle. Diffused aeration provided complete mixing and aeration. The culture was fed two times per day approximately 1000 mg/L ammonia-nitrogen in an inorganic nutrient solution buffered by sodium bicarbonate. The hydraulic retention time equalled the solids retention time of 25 days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
24 h
Test temperature:
25 °C
pH:
6.5 to 8
Details on test conditions:
The inhibition of ammonia consumption was used as the criterion for toxic inhibition of Nitrosomonas. Sealed serum bottles were prepared with ammonia feed and 20 mL of surcharged oxygen. Ammonia was measured at the end of the assay period (24 hours) using an ammonia selective electrode. Nitrite was checked to ensure that only toxicity to Nitrosomonas and not toxicity to Nitrobacter was controlling the rate of metabolic activity.
Reference substance (positive control):
not required
Remarks:
A set of 82 substances was tested.
Duration:
24 h
Dose descriptor:
IC50
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Validity criteria fulfilled:
not applicable
Conclusions:
The ammonia consumption of Nitrosomonas was inhibited by 50 % at a concentration of TCP in water of 30 mg/L
Executive summary:

A static test on toxicity of TCP to Nitrosomonas was carried out at 25 °C using activated sludge from a plant treating meat-packing, rendering and hide-curing wastewater. The criterion for toxicity to Nitrosomonas was the inhibition of ammonia consumption. After 24 hours it was found that ammonia consumption was inhibited by 50 % at a TCP concentration of 30 mg/L , which was taken as the IC50 value.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was carried out following principles that are documented in sufficient detail in the publication to consider the experimental results to be reliable with restrictions.
Qualifier:
equivalent or similar to guideline
Guideline:
other: Vibrio fischeri (MICROTOX® test) ISO 11348-1, -2, -3 (1999)
Principles of method if other than guideline:
Testing was performed using a Microtox® model 2055 toxicity analyzer and standard procedures recommended by Microbics Corporation.
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,2,3-Trichloropropane
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Photobacterium phosphoreum
Details on inoculum:
reconstituted freeze-dried Photobacterium phosphoreum (Microtox® bacteria), which has been renamed to Vibrio fischeri
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5 h
Details on test conditions:
Microtox® model 2055 toxicity analyzer
TEST SYSTEM
- Test vessel: Type open
EFFECT PARAMETERS MEASURED: bioluminescence inhibition
Reference substance (positive control):
not required
Remarks:
A set of 83 chemicals was tested.
Duration:
5 min
Dose descriptor:
IC50
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of bioluminescence
Validity criteria fulfilled:
not applicable
Conclusions:
50% reduction in bioluminescence of Photobacterium phosphoreum as observed at a TCP concentration of 19 mg/L.
Executive summary:

Testing was performed using a Microtox® model 2055 toxicity analyzer and standard procedures recommended by Microbics Corporation. The test was performed using instrumentation and supplies from the Microbics Corporation. No GLP conditions or officially recognized guidelines applied.

The test is based in the bioluminescence of reconstituted freeze-dried Microtox® bacteria (Photobacterium phosphoreum) as a measure of biological activity. Light emission by bacteria is decreased by the addition of toxicants. Five minutes test results were used.

A TCP concentration of 19 mg/L in the test medium produced a reduction of 50% biolumiescence and is accordingly regarded as IC50 concentration.

Description of key information

TCP exhibits acute effects on aquatic micro-organisms.

Key value for chemical safety assessment

EC50 for microorganisms:
290 mg/L

Additional information

Blum & Speece (1991) report results from four different assays employing micro-organism communities and specific strains. However methanogens were most susceptible with regard to the endpoint inhibition of gas production (48-h IC50 0.63 mg/L), the aerobic heterotrophic community with the endpoint inhibition of oxygen uptake represent the relevant community for assessment. The study protocol is comparable to OECD TGD 209 and the study is considered valid and conclusive and yielded a 24-h IC50 of 290 mg/L.

The assay employing nitrifying bacteria (Nitrosomonas sp.) with the endpoint inhibition of ammonia consumption revealed an 24-h IC50 of 30 mg/L. This is in agreement with ECHA (2008, information requirements and chemical safety assessment, chapter R.10., p 30) who recommends an assessment factor (AF) of 10 to be applied to result of a sludge respiration test, reflecting the lower sensitivity of this endpoint as compared to nitrification.

Finally a Microtox® model 2055 assay (ISO 11348) employing a specific bacterial population (Vibrio fischeri) with the endpoint inhibition of bioluminescence showed an 5-min IC50 value of 19 mg/L.