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Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
Please refer also to the read-across statement attached in section 13

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The target and the source substances are structurally similar substances that share the common organometallic core structure consisting of a central zinc metal bonded to four alkyldithiophosphate esters (ligands) by coordinate covalent bonds -Zn[(S2P(OR)2]2. Structural variations between the target and the source substances are related only to the alkyl (R) groups of the alkyldithiophosphate ligands. The substances in this category give thus rise to an (identical) common compound Phosphorodithioic acid moiety that can be released by the breakage of ester bonds and dissociation from the Zinc complex to which the organism would be exposed if the target substance was tested in the toxicity studies. Exposure to the parent compounds (non-transformed constituents) and to the counter alkyl alcohols, possibly released by hydrolysis of P-O bonds – non-common compounds – would not influence the prediction of the (eco)toxicological properties because they are considered to have the same biological targets and to cause the same type of effects through a common underlying mechanism due to the same functional groups (zinc cation, phosphorodithioic cation and aliphatic alcohol anionic moieties). The impurities of the target and the source substances are not expected to impact the prediction because they are identical or, if slightly structural different, belong to the same class of compounds with the same functional groups and their percentages are very low.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
The aquatic toxicity of the ZDDP category members is mainly influenced by the molecular weight (depends on the length of alkyl rests), and the water solubility. Numerous studies demonstrate decreasing toxicity with the increased chain length. Since the alkyl rests in the target substance are shorter (isobutyl, isopropyl and pentyl) than that of the source substance CAS 4259-15-8 (ethylhexyl) and the water solubility is higher (40.7 mg/L vs 9.1 mg/L for the target and the source substance, respectively) a higher bioavailability is expected for the target substance when compared to source substance CAS 4259-15-8. Regarding the source substance CAS 84605-29-8, the water solubility is with 2764 mg/L above the one of the target substance. Therefore, this source substance is expected to be more bioavailable than the target substance. Thus, data on CAS 84605-29-8 can serve as worst-case data for the target substance. In general, intrinsic toxicities of substances may be the same, because they have the same functional groups and thus the same mode of action. Therefore, taking into account the similar predicted environmental fate and behaviour, the target substance is considered not to possess significantly higher toxicity potential to aquatic environment then the source substance. Thus, the results of the toxicity studies in microorganisms available for the source substances are considered to be similar if these studies were conducted with the target substance. However, the target substance should be classified as aquatic toxic with long-lasting effects because its alkyl chains lengths are shorter (< C8) and thus aquatic toxicity potential cannot be ruled out.
GLP compliance:
yes
Details on inoculum:
Activated sludge was obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool. One gallon of activated sludge was collected by the plant operator, picked up on the day of collection and transported to the testing laboratory.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The average respiration rates for the two controls was 77.2 mg O2/L*hr.

The resultant consumption rates for the inoculum exposed to the reference toxicant were used to calculate the EC50 of the reference toxicant in the sludge used to test the experimental material. The EC50 was calculated to be 18.5 mg/L 3,5-dichlorophenol.

Five nominal concentrations were tested with the activated sludge inoculum at 1, 10, 100, 1000 and 10000 mg/L. The resultant respiration rates were respectively: 67.0, 58.2, 67.2, 72.0 and 54.0 mg O2/L*hr. These data translate into percent inhibitions of 13.2%, 24.6%, 13.0%, 6.7% and 30.1%. Because none of the test concentrations produced an inhibition greater than 50%, the EC50 was estimated to be > 10,000 mg/L.
Results with reference substance (positive control):
Results with reference substance valid? Yes.

To assure quality control, the reference toxicant 3,5-dichlorophenol needs to be between 5 and 30 mg/L. The EC50 for the reference toxicant in this test was calculated to be 18.5 mg 3,5-dichlorophenol per liter.

Test Material Data

Test Concentration (mg/L)

Change in O2 (mg/L)

Change in Time

(minutes)

O2

Consumption

(mg/L*hr)

Percent Inhibition

1

4.1

3 4/6

67

13.2

10

5.5

5 4/6

58.2

24.6

100

5.6

5

67.2

13.0

1,000

4.2

3 3/6

72

6.7

10,000

7.5

8 2/6

54

30.1

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms using standard guidelines gave an EC50 of >10,000 mg/L.
Executive summary:

Introduction:

A study was performed under GLP to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984), No. 209 "Activated Sludge, Respiration Inhibition Test".

Methods:

Activated sewage sludge was exposed to directly added test material at concentrations of 1, 10, 100, 1000 and 10000 ppm, a duplicate control group, and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant 3,5-dichlorophenol. The test duration was a 3 hour exposure period at 20 °C to the test material followed by measurement of oxygen consumption.

 

Results:

The effect of the test material on the respiration of activated sewage sludge gave an EC50 of >10,000 mg/L. The reference material gave an EC50 value of 18.5 mg/L. The activated sludge respiration test passed the quality control criteria for an acceptable test. The two control replicates produced oxygen consumption rates within the required 15% of each other.

Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida growth inhibition test
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
Please refer also to the read-across statement attached in section 13

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The target and the source substances are structurally similar substances that share the common organometallic core structure consisting of a central zinc metal bonded to four alkyldithiophosphate esters (ligands) by coordinate covalent bonds -Zn[(S2P(OR)2]2. Structural variations between the target and the source substances are related only to the alkyl (R) groups of the alkyldithiophosphate ligands. The substances in this category give thus rise to an (identical) common compound Phosphorodithioic acid moiety that can be released by the breakage of ester bonds and dissociation from the Zinc complex to which the organism would be exposed if the target substance was tested in the toxicity studies. Exposure to the parent compounds (non-transformed constituents) and to the counter alkyl alcohols, possibly released by hydrolysis of P-O bonds – non-common compounds – would not influence the prediction of the (eco)toxicological properties because they are considered to have the same biological targets and to cause the same type of effects through a common underlying mechanism due to the same functional groups (zinc cation, phosphorodithioic cation and aliphatic alcohol anionic moieties). The impurities of the target and the source substances are not expected to impact the prediction because they are identical or, if slightly structural different, belong to the same class of compounds with the same functional groups and their percentages are very low.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
The aquatic toxicity of the ZDDP category members is mainly influenced by the molecular weight (depends on the length of alkyl rests), and the water solubility. Numerous studies demonstrate decreasing toxicity with the increased chain length. Since the alkyl rests in the target substance are shorter (isobutyl, isopropyl and pentyl) than that of the source substance CAS 4259-15-8 (ethylhexyl) and the water solubility is higher (40.7 mg/L vs 9.1 mg/L for the target and the source substance, respectively) a higher bioavailability is expected for the target substance when compared to source substance CAS 4259-15-8. Regarding the source substance CAS 84605-29-8, the water solubility is with 2764 mg/L above the one of the target substance. Therefore, this source substance is expected to be more bioavailable than the target substance. Thus, data on CAS 84605-29-8 can serve as worst-case data for the target substance. In general, intrinsic toxicities of substances may be the same, because they have the same functional groups and thus the same mode of action. Therefore, taking into account the similar predicted environmental fate and behaviour, the target substance is considered not to possess significantly higher toxicity potential to aquatic environment then the source substance. Thus, the results of the toxicity studies in microorganisms available for the source substances are considered to be similar if these studies were conducted with the target substance. However, the target substance should be classified as aquatic toxic with long-lasting effects because its alkyl chains lengths are shorter (< C8) and thus aquatic toxicity potential cannot be ruled out.
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
380 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
EC0
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Results with reference substance (positive control):
Results with reference substance valid? yes
- Relevant effect levels: EC50 = 19 mg/L
- Other: EC10 = 13 mg/L
Reported statistics and error estimates:
ECx values were determined by graphic evaluation

Optical density measurements

a) test substance

Test substance concentration (mg/L)

Dilution series factor (f=2) of stock solution

Optical density (436 nm)

1

2

3

469

2

0.53

0.584

0.548

235

4

1.54

1.61

1.57

118

8

1.59

1.57

1.63

59

16

1.64

1.68

1.65

b) control

Control

Optical density (436 nm)

1

1.60

 

2

1.58

 

3

1.35

 

4

1.62

 

5

1.58

 

6

1.61

 

7

1.59

 

8

1.52

 

mean

1.56

Growth inhibition

Test substance concentration (mg/L)

FNU (436 nm)

Inhibition (%)

1

2

3

mean

469

133

146

137

139

64

235

385

403

393

327

16

118

398

393

408

400

0

59

410

420

413

414

0

Control

400, 395, 338, 405, 395, 403, 398, 380

389

 

Validity criteria fulfilled:
yes
Remarks:
The test is considered as valid as the validity criteria were met (multiplication factor of the inoculum in the controls > 60, EC50 of the reference substance 3,5 -dichlorophenol 10-30 mg/L).
Conclusions:
The effect of the test material on growth inhibition of Pseudomonas putida leads to an EC50 of 380 mg/L.
Executive summary:

A study was performed under GLP to assess the effect of the test material on the growth inhibition of Pseudomonas putida. The method followed that described in the DIN EN ISO 10712 Pseudomonas putida growth inhibition test.

The effect of the test material on growth inhibition of Pseudomonas putida leads to an EC50 of 380 mg/L and an EC0 of 200 mg/L.

Description of key information

Based on the different approaches of the two tests (respiration vs. growth inhibition) both studies are seen as key studies:

Source substance Phosphorodithioic acid, mixed O,O-bis(1,3 -dimethylbutyl and iso-Pr) esters, zinc salts (CAS 84605 -29 -8):

A study was performed under GLP to assess the effect of the test material on the respiration of activated sewage sludge (Klimisch 2). The method followed that described in the OECD Guidelines for Testing of Chemicals (1984), No. 209 "Activated Sludge, Respiration Inhibition Test".

The effect of the test material on the respiration of activated sewage sludge micro-organisms using standard guidelines gave an EC50 of >10,000 mg/L.

Source substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259 -15 -8):

A study was performed under GLP to assess the effect of the test material on the growth inhibition of Pseudomonas putida (Klimisch 1). The method followed that described in the DIN EN ISO 10712 Pseudomonas putida growth inhibition test.

The effect of the test material on growth inhibition of Pseudomonas putida leads to an EC50 of 380 mg/L and an EC0 of 200 mg/L.

For CSA the lower value is choosen, also with regard, that growth inhibition is an earlier stage than respiration inhibition.

Key value for chemical safety assessment

EC50 for microorganisms:
380 mg/L
EC10 or NOEC for microorganisms:
200 mg/L

Additional information

Phosphorodithioic acid, mixed O,O-bis(1,3 -dimethylbutyl and iso-Pr) esters, zinc salts (CAS 84605 -29 -8):

Introduction:

A study was performed under GLP to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984), No. 209 "Activated Sludge, Respiration Inhibition Test".

Methods:

Activated sewage sludge was exposed to directly added test material at concentrations of 1, 10, 100, 1000 and 10000 ppm, a duplicate control group, and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant 3,5-dichlorophenol. The test duration was a 3 hour exposure period at 20 °C to the test material followed by measurement of oxygen consumption.

 Results:

The effect of the test material on the respiration of activated sewage sludge gave an EC50 of >10,000 mg/L. The reference material gave an EC50 value of 18.5 mg/L. The activated sludge respiration test passed the quality control criteria for an acceptable test. The two control replicates produced oxygen consumption rates within the required 15% of each other.

Source substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259 -15 -8):

A study was performed under GLP to assess the effect of the test material on the growth inhibition of Pseudomonas putida. The method followed that described in theDIN EN ISO 10712 Pseudomonas putida growth inhibition test.

The effect of the test material on growth inhibition of Pseudomonas putida leads to an EC50 of 380 mg/L and an EC0 of 200 mg/L.


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