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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 01, 1991 - Jun 03, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
The study was performed acording to a method similar to the one described in OECD guideline No. 209 and followed the principles of GLP. However, relatively few details are provided in the study documentation.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
1986
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
no details provided
Test organisms (species):
activated sludge
Details on inoculum:
Laboratory culture: The activated sludge was obtained on 29.05.1992 from a laboratory water treatment plant (OECD).
Pretreatment: None
Initial biomass concentration: 6 g/L dry weight
Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
nominal concentrations: 100, 180, 320, 560, and 1000 mg/L
Details on test conditions:
Autoxidation was determined as 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
237 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Please, see section "Any other information on results incl. tables" for the values for the inhibition of activated sludge respiration observed with different concentrations of the test item and the attached image below for a graphical presentation of the results in an inhibition vs. concentration-diagram.

Table 1: Results of the activated sludged respiration inhibition assay

Test concentration (mg/L)

Respiration rate (mg/L x h)

Inhibition (%)

100

28.3

18.7

180

20.3

41.7

320

11.3

67.5

560

7.0

79.9

1000

6.0

82.8

Validity criteria fulfilled:
yes
Remarks:
Validity of the test system was checked by a graphical evaluation of the results obtained with the reference substance.
Conclusions:
The effective concentration 50% (EC50) for inhibition of activated sludge respiration was determined as 237 mg/L. In view of this low bacteriotoxic activity, it was concluded that release of the test compound into the environment would most likely not adversely affect the natural self purification capacity of surface water bodies.
Executive summary:

The toxicity of the test item, Menthol H&R Komp., to microorganisms was tested in an activated sludge respiration assay. This assay was performed according to the ISO guideline 8192 -1986 (E), which largely resembles the OECD guideline No. 209. In this assay, The effective concentration 50% (EC50) for inhibition of activated sludge respiration by the test item was determined as 237 mg/L.

Since this value indicates a rather low bacteriotoxic activity, it was concluded that release of the test compound into the environment would most likely not adversely affect the natural self purification capacity of surface water bodies.

The validity of the test system was checked by testing a confirmed bacteriotoxic compound, 3,5-dichlorophenol, as reference substance.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Dec 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Test organisms (species):
activated sludge
Details on inoculum:
- Initial biomass concentration: 6.0 g/L (dry weight)
- laboratory scale sewage treatment plant treating synthetic sewage (OECD 209)


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.8 °C
pH:
8.0 - 8.3
Dissolved oxygen:
O2 - Start: 5.2 - 8.4 mg
O2 - End: 3.0 - 8.2 mg
Nominal and measured concentrations:
nominal concentrations: (preliminary Test) 100, 1000 and 10000 mg/L
nominal concentrations: (primary test) 100, 180, 320, 560 and 1000 mg/L

Details on test conditions:
Type: aquatic
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorphenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
306 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The concentrations of the test item and reference compound were not confirmed by analytical methods, but instead based on nominal concentrations
Results with reference substance (positive control):
EC50 of the reference compound 3.5-Dichlorophenol is < 20 mglL

Results:

- preliminary Test:

 Test conc. [mg/l]  Respirations rate [mg/l*h]  Inhibition [%]  Referenz substance [mg/l]   Respirations rate [mg/l*h]   Inhibition [%]
 100  22  0  1.0  22  0
 1000  3  85  20  5  75
 10000  0  100      

Results:

- primary Test:

  Test conc. [mg/l]   Respirations rate [mg/l*h]   Inhibition [%]   Referenz substance [mg/l]   Respirations rate [mg/l*h]   Inhibition [%]
 100  19  7  1.0  22  0
 180  17  19  20  5  75
 320  7  67      
 560  4  80      
 1000  2  88      
Validity criteria fulfilled:
yes
Remarks:
on the basic of graphic evaluation; The EC50 for 3,5-DCP was 20 mg/L and between the prescribed limits of 5 and 30 mg/L.
Conclusions:
For DL-Menthol (CAS no. 1490-04-6) an EC50 (3 h) of 306 mg/L was observed.
Executive summary:

A study with activated sludge with a duration fo 3 hours was performed according to the OECD Guideline 209. For DL-Menthol (CAS no. 1490-04-6) an EC50 of 306 mg/L was observed.

Description of key information

Activated sludge respiration inhibition EC50 (3 h) = 237 mg/L (nominal); EC10 (3 h) = 51 mg/L (nominal); read-across

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
237 mg/L
EC10, LC10 or NOEC for microorganisms:
51 mg/L

Additional information

Since no data for the target substance are available, the toxicity to activated sludge microorganisms is assessed on the basis of data from two structurally similar menthols (stereo isomers).
The toxicity to microorganisms was investigated for L-menthol (CAS 2216-51-5) and an unspecified menthol isomer mixture (D/L-menthol, CAS 1490-04-6). The studies with both source substances investigated the respiration inhibition of activated sludge microorganism under standard test conditions in accordance to GLP.
In the key study with L-menthol (CAS 2216-51-5), which was conducted according to ISO 8192 (similar to OECD 209), an EC50 of 237 mg/L (nominal) and an EC10 of 51 mg/L (nominal) were determined after 3 h based on inhibition of respiration. The test system was validated using 3,5-dichlorophenol as reference substance.
This result was confirmed in the supporting study performed with the source substance D/L-menthol (CAS 1490-04-6). This study was conducted according to OECD 209 (GLP). An EC50 of 306 mg/L (nominal) was observed after 3 h of exposure.
Based on the results from two structurally related source substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that testing the target substance would result in comparable results.