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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-05-19 - 1992-05-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study, limited details on study description available
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
GLP compliance:
yes
Remarks:
No GLP certificate available.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
No surrogate or analogue material was used.
Analytical monitoring:
no
Details on sampling:
No details available.
Vehicle:
no
Details on test solutions:
No details available.
Test organisms (species):
activated sludge
Details on inoculum:
- Source: sewage treatment plant of laboratory (OECD)
- Sample taking: 1992-05-15
- Pretreatment: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
Not mentioned in the test report, only in the applied guideline.
Post exposure observation period:
No post exposure observation period described.
Hardness:
No details available.
Test temperature:
No details available.
pH:
No details available.
Dissolved oxygen:
No details available.
Salinity:
Not applicable.
Nominal and measured concentrations:
56, 100, 180, 320 and 560 mg/L
Details on test conditions:
TEST SYSTEM
- Biomass loading rate: 6 g/L TS
- Autooxidation: 10000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenole
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
383 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
56 mg/L: respiration rate of 19.7 mg/L x h, corresponding to an inhibition of 0 %
100 mg/L: respiration rate of 17.1 mg/L x h, corresponding to an inhibition of 10 %
180 mg/L: respiration rate of 14. 0 mg/L x h, corresponding to an inhibition of 26.3 %
320 mg/L: respiration rate of 11.0 mg/L x h, corresponding to an inhibition of 42.1 %
560 mg/L: respiration rate of 7.0 mg/L x h, corresponding to an inhibition of 63.2 %
Results with reference substance (positive control):
No details available.
Reported statistics and error estimates:
No details available.

Table 1: Overview of results

Test concentration

[mg/L]

Respiration rate [mg/L x h]

Inhibition

[%]

56

19.7

0.0

100

17.1

10.0

180

14.0

26.3

320

11.0

42.1

560

7.0

63.2

Figure 1: Overview of results

Please find attached.

Validity criteria fulfilled:
yes
Remarks:
Basic information available.
Conclusions:
The study is regarded as a guideline study with GLP compliance, however, only basic information are reported. The outcome of the experiment can be considered as for the chemical safety assessment as well as for the risk assessment, since all validity criteria of the test method were met. Based on the obtained EC50 (383 mg/L), the substance is concerned as not toxic towards aquatic microorganisms in sewage treatment plants.
Executive summary:

The toxicity of 4-phenylbutenone to aquatic microorganisms was investigated according to ISO 8192, which corresponds in most parts to OECD Guideline 209 (Kanne, 1993). A defined volume of activated sludge is mixed with synthetic culture medium and incubated to investigate the respiration rate. This respiration rate is compared to inoculums with different test substance respiration rates. 3,5 -Dichlorphenol is used as reference compound. The activated sludge was not pretreated before. Test concentrations of 56, 100, 180, 320 and 560 mg/L were used. As result, an EC50 of 383 mg/L was reported, which corresponds to a nominal concentration since no analytical monitoring was conducted.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Well-documented publication about Microtox testing with marine luminescence bacteria
Qualifier:
according to guideline
Guideline:
other: Microtox testing with Photobacterium phosphoreum (marine luminescent bacterium)
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
No surrogate or analogue material was used.
Analytical monitoring:
yes
Details on sampling:
Flow-proportional wastewater samples were taken at five different episodes in 1989. Each episode lasted two weeks and included fourteen 24 h samples. During the sampling period the wastewater flow was registered. The samples were collected and immediately frozen (-20 °C). Five two-week samples were composed by flow-proportional mixing of fourteen 24 h subsamples representing each sampling period. A mixed sample was composed by flow-proportional mixing of seventy 24 h subsamples.
Vehicle:
yes
Details on test solutions:
Three different test levels were included with different test solutions:
1) A set of chemical analysis and a Microtox test on the two-week samples
2) An extensive test program of the mixed sample
3) A set of tests on the degraded mixed sample
For benzylidene acetone the saturated water phase was used for the Microtox test (48 h, 25 °C).
Test organisms (species):
other: Photobacterium phosphoreum for Microtox testing of the test substance, activated sludge for respiration and nitrification inhibition of the sample mixture
Details on inoculum:
Marine luminescence bacteria: Photobacterium phosphoreum
Test type:
static
Water media type:
saltwater
Total exposure duration:
15 min
Remarks on exposure duration:
Mikrotox testing: 15 min, Inhibition of respiration and nitrification of mixture: 4 h
Post exposure observation period:
No post exposure observation period described.
Hardness:
No details available.
Test temperature:
No details available.
pH:
7.0 - 8.0 (adjusted)
Dissolved oxygen:
No details available.
Salinity:
2 % NaCl (Microtox Testing)
Nominal and measured concentrations:
0, 1, 10 and 50 % (v/v) for respiration inhibition test
0, 1, 10 and 40 % (v/v) for nitrification inhibition test
Details on test conditions:
The Microtox test was performed as described by the International Organization for Standardization for an exposure time of 15 min with the test substance.
The inhibition of respiration of activated sludge was investigated according to Danish standard DS 297 over an exposure period of 4 h.
The inhibition of nitrification of activated sludge was investigated according to draft international standard ISO/DIS 9509 for an exposure period of 4 h.
Reference substance (positive control):
not required
Remarks:
Different chemicals were tested.
Duration:
15 min
Dose descriptor:
EC10
Effect conc.:
0.03 other: %
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Concentration at which the luminosity of the bacteria decreases by 10 %.
Duration:
15 min
Dose descriptor:
EC50
Effect conc.:
0.21 other: %
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Concentration at which the luminosity of the bacteria decreases by 50 %.
Details on results:
Benzylidene acetone shows a high toxicity towards the luminescent bacteria.
Results with reference substance (positive control):
Not applicable. Different chemicals were tested.
Reported statistics and error estimates:
For calculations the Microtox Version 5.20 program was used.
Validity criteria fulfilled:
yes
Remarks:
Test procedure according to national Guidelines.
Conclusions:
The publication describes a valid guideline study without information about GLP compliance. According to the reported results the test substance showed high toxicity with the luminescent bacteria.
Executive summary:

Brorson et al. (1994) investigated the potential environmental impact of wastewater from a chemical-pharmaceutical plant. For this purpose, the test substance was investigated in a mixture for its potential to inhibit nitrification (according to ISO/DIS 9509) and respiration (according to Danish standard DS297). Furthermore, a Microtox test was conducted, whereby marine luminescence bacteria Photobacterium phosphoreum was exposed for 15 minutes to the test substance and the resulting EC50 value of 0.21 % corresponds to the concentration at which the luminosity of the bacteria decreases by 50 %. According to this, the test substance showed low toxicity towards the bacteria.

Description of key information

OECD 209, activated sludge: EC50 = 383 mg/L
Microtox test with Photobacterium phosphoreum: EC50(15min) = 0.21 %  (2100 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:
383 mg/L

Additional information

The toxicity of 4-phenylbutenone towards aquatic microorganisms was investigated according to ISO 8192, which corresponds in most parts to OECD Guideline 209 (Kanne, 1993). A defined volume of activated sludge is mixed with synthetic culture medium and incubated. The respiration rate was measured during this time and compared to inoculums with different test substance respiration rates. 3,5-Dichlorphenole is used as reference compound. The activated sludge was not pretreated before. Test concentrations of 56, 100, 180, 320 and 560 mg/L were used. As result, an EC50 of 383 mg/L was reported, which corresponds to a nominal concentration since no analytical monitoring was conducted.

Further information is given as followed: Brorson et al. (1994) investigated the potential environmental impact of wastewater from a chemical-pharmaceutical plant. For this purpose, the test substance was investigated in a mixture for its potential to inhibit nitrification (according to ISO/DIS 9509) and respiration (according to Danish standard DS297). Furthermore, a Microtox test was conducted, whereby marine luminescence bacteria Photobacterium phosphoreum was exposed for 15 minutes to the test substance and the resulting EC50 value of 0.21 % corresponds to the concentration at which the luminosity of the bacteria decreases by 50 %.