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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non-GLP
Qualifier:
according to guideline
Guideline:
other: DIN 38412 Part 27
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Heliogen Gruen 8730
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tween 80. Tween 80 (100 mg/l) has been used as emulgator. The test solution was stirred for 17 h at 25 °C.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: yes
- Method of cultivation: The stemculture is taken up in 0.9 % strength NaCl solution. Batches of 200 ml of culture fluid (25 g/l of nutrient broth I) are inoculated with cell suspension under sterile conditions.
- Preparation of inoculum for exposure: The cells are harvested after 16 ± 2 h of shaking on a rotary shaker at 22 ± 2 °C and 200 r.p.m. The cells harvested are separated by centrifugation for 10 min at 6000 r.p.m. and at 10 °C in a refrigerated centrifuge, washed twice with phosphate buffer and resuspended in buffer. Absorbence measurements are carried out in 1 cm cells on an aliquot of the cell suspension at a dilution of 1:100. A value of 200 TE/F is set at a wavelength of 436 nm against the buffer blank value.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Test temperature:
24 ± 1 °C
pH:
7.6-8.4
Nominal and measured concentrations:
Nominal test concentration:
control, 5000 mg/l, 10000 mg/l, 10000 mg/l without glucose, solvent control (Tween80)
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 25 g/l Nutrient broth / 18 g/l Agar poured into slant-agar tube. 5 ml of glucose (198 g/l), 95 ml of the test substance, 2-3 ml of bacterial suspension
Key result
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
30 min
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
Duration:
30 min
Dose descriptor:
other: EC90
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Oxygen consumption of Pseudomonas putida after 30 min:
***********************************************************
* nominal       *     OXYGEN CONSUMPTION       *          *
* concentration * mean value   *  % of control * pH-value *
* [mg/l]        * (mg/l * min) *               * test mix *
***********************************************************
*    control    *    2.575     *      100      *          *
*     10 000    * > control    * > control     *    8.4   *
*      5 000    *      2.6     *      101      *    7.6   *
*  wG 10 000    *        0     *        0      *          *
*    Tween80    *     2.45     *     95.1      *          *
***********************************************************
wG = without glucose,   Tween80 = solvent control
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Pigmatex Blue 3G 60432
- Storage conditions: In original container at room temperature in darkness
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An aqueous dispersion was prepared using ethylene gylcol was prepared.
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: A mixed population of activated sewage sludge micro-organisms.
- Name and location of sewage treatment plant where inoculum was collected: The aeration stage of the HRC Ltd. sewage treatment plant, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample was maintained at 22 ± 2°C with continuous aeration and used on the day of collection. The pH was buffered to pH 7.6 with sodium bicarbonate solution and the suspended solids level adjusted to 3.6 g/l prior to use
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
30 min
Remarks on exposure duration:
and 3 h
Hardness:
350 mg/l CaCO3
Test temperature:
22 +/- 2°C
Nominal and measured concentrations:
Nominal test concentration: 10000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks (500 ml)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water, dechlorinated by sodium thiosulphate
- Photoperiod: Normal laboratory lighting.
Reference substance (positive control):
yes
Remarks:
3,5 diclorphenol
Key result
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
Duration:
30 min
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
Results with reference substance (positive control):
30-min EC50: 16 mg/L
3-h EC50: 12 mg/L

Table 1. Oxygen consumption rates and percentage inhibition for 30 minutes contact time.

Concentration [mg/L]

O2consuptionrates [mgO2/L/min]

Inhibition [%]

Control 1

0.56

-

Control 2

0.58

-

TS 1 1000

1.36

- 139 *

TS 2 1000

1.36

- 139 *

RS 3.2

0.52

8.8

RS 10

0.41

28

RS 32

0.14

75

* Increase

 

Table 2. Oxygen consumption rates and percentage inhibition for 3 hours contact time.

Concentration [mg/L]

O2consuptionrates [mgO2/L/min]

Inhibition [%]

Control 1

0.54

-

Control 2

0.57

-

TS 1 1000

0.59

- 6 *

TS 2 1000

0.61

- 10 *

RS 3.2

0.48

14

RS 10

0.30

46

RS 32

0.12

78

* Increase

TS – test substance

RS – reference substance

 

The increase in oxygen consumption rates of the two test substance replicates over the controls are probably due to the ethylene glycol in the formulation acting as an additional substrate

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:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
non-GLP study
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
Draft
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Kupferphthalocyanin,
- Analytical purity: 99.3 %
Analytical monitoring:
no
Details on test solutions:
Substance is hardly soluble in water and were directly added without pretreatment.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: Activated sludge from a laboratory wastewater plant treating municipal sewage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Test temperature:
20 - 25 °C
Nominal and measured concentrations:
Nominal test concentration 500 and 1000 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
ca. 750 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Nominal test concentration [mg/L]

Respiration rate [mg/l*h]

Respiration inhibition [%]

Respiration promotion [%]

500

30

0

7

1000

16

43

0

Description of key information

The substance is not toxic to aquatic microorganisms, with an EC50 > 10000 mg/L and an EC10 > 10000 mg/L.

Key value for chemical safety assessment

Additional information

All substances in this category share a similar structure with a molecular weight > 500 g/mol. Based on the high molecular weight and insolubility, the substances in this category will not be able to penetrate biological membranes. Thus, no systemic exposure is expected. This is confirmed by the environmental studies on microorganisms for two substances in this category: CAS 1328-53-6 and CAS 147-14-8. The environmental toxicity data can be used for read-across to all substances in this category.


In a study conducted according to ISO 8192 with domestic activated sludge, the 30-min EC20 of substance CAS 147-14-8 for inhibition of total respiration was determined to be ca. 750 mg/L (BASF 1988, K2). The 3-h EC50 was found to be > 10000 mg/L in the second study with activated sludge conducted according to OECD TG 209 (Huntington Research Centre 1988, K2). Additional experimental data are available for the substance CAS 1328-53-6. In a study with Pseudomonas putida according to DIN 38412-8, the 30-min EC10 of CAS 1328-53-6 for inhibition of total respiration was determined to be > 10000 mg/L (BASF 1988, K2).


The EC10, EC20 and EC50 were significantly higher than the water solubility of the test substance. Based on available information, it is concluded that all substances in the category were not toxic to aquatic microorganisms with an EC50 > 10000 mg/L and an EC10 > 10000 mg/L.