Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 october 2012 - 6 february 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to an international guideline and according to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTIONS
- Preparation of test item solutions :
A stock solution with a nominal concentration of 13.81 mg test item/L was prepared by dissolving 13810 mg of the test item in 1000 mL water and thoroughly mixed by intense stirring for 10 minutes. In this way a clear solution was obtained. The pH of the stock solution was adjusted from 8.3 to 7.4 with 0.5M sulfuric acid solution. Adequate volumes of this stock solution were diluted with water to prepare the test media with the lower test item concentrations. The exact amounts used are given in the table1. Then, 16 mL of synthetic sewage feed and 250 mL of activated sludge inoculum were added.

- Preparation of reference substance (3,5-dichlorophenol) solutions :
A stock solution of 3,5-dichlorophenol was prepared by dissolving 0.5g of 3,5-dichlorophenol directly in about 950 mL water with the aid of ultrasonification and intense stirring. The 3,5-dichlorophenol solution was adjusted with NaOH to pH 7.7 and make up to one liter. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with water, synthetic sewage feed and inoculum in the respective vesssels to obtain the desired test concentrations (3.2, 10 and 32 mg/L).

- Preparation of test system :
At the start of the test, 16 ml of synthetic sewage were made up to 250 mL with water and 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to the first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage were added to the appropriate amounts of reference item or test item in water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum.
The addition of the inoculum to the incubation mixture is considered as the start of the 3-hour incubation period.

- Evidence of undissolved material (e.g. precipitate, surface film, etc) : no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of the inoculum : aerobic sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.
- Pretreatment : no
- Method of cultivation : no data
- Preparation of inoculum for exposure : The sludge was washed twice with the chlorine free tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and reajusted to the level of 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge was 7.9.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Test temperature:
The temperature in the test media, measured in one control, was 20°C at the start and at the end of the incubation period.
pH:
7.4 - 7.6 (test substance)
7.3 - 8.0 (reference substance)
Dissolved oxygen:
3.6 mgO2/l - 1.8 mgO2/l (test substance)
6.5 mgO2/l - 2.4 mgO2/l (reference substance)
Nominal and measured concentrations:
173, 345, 691, 1381 and 2762 mg test item/L corresponding to 62.5, 125, 250, 500 and 1000 mg active ingredient/L
3.2-10 and 32 mg/l (reference substance)
Details on test conditions:
TEST SYSTEM
- Test vessel
- Type : Open
- Material, size, headspace, fill volume : the test was performed in 2000 mL glass beakers containing 500 mL of incubation mixture
- Aeration :during the incubation period of 3 hours, all vessels were aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.
- No. of vessels per concentration (replicates) : threee flasks per concentration (test item) - one flask per concentration (reference item) - six blank controls (containing an equal volume of activated sludge and synthetic medium but no test or reference item)
- The vessels were prepared in the following order : two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and then two blank control vessels.

TEST MEDIUM / WATER PARAMETERS : activated sludge with the addition of a synthetic sewage
- source / preparation of dilution water : the test water used for the test was laboratory tap water dechlorinated by passage through an activated carbon filter and partly softened giving water with a total hardness of approximately 140 mg/l as CACO3.

- Synthetic sewage feed composition :
Peptone : 16 g
Meat extract : 11 g
Urea : 3 g
NaCl : 0.7 g
CaCl2.2H2O : 0.4 g
MgSO4.7H2O : 0.2 g
K2HPO4 : 2.8 g
dissolved in 1 litre of deionized water. The pH of this solution was 7.2.

EFFECT PARAMETERS MEASURED
For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the oxygen consumption rate (in mg O2 L-1 minute-1) was measured with an oxygene electrode and was continuously recording. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate was determined from the linear part of the respiration curve normally in the range 7-2.5 mg O2/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Acros Organics BVBA; Batch : A0257431) - purity : 99% (GC).Expiration date : 13 march 2013
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 2 762 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 328 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solid content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
At all tested concentrations up to and including 2762 mg test item/L, corresponding to 1000 mg active ingredient/L, the inhibition of the respiration rate of activated sludge after the incubation period of three hours was in the range -56.7 to 1.2% compared to the control, i.e. a stimulatory effect was observed for all tested concentrations except the highest one.
According to these results, the test item had no significant inhibitory effect on the respiration rate of up to and including the highest test item concentration of 2762 mg/L.

Thus, the 3-hour NOEC of test item to activated sludge microorganisms was at least 2762 mg/L (1000 mg active ingredient/L). This value might even be higher but concentrations above 2762 mg/L (1000 mg active ingredient/L) were not tested. The 3-hour EC20, EC50 and EC80 could not be calculated but were clearly higher than 2762 mg/L (1000 mg active ingredient/L).

The specific respiration rates for the two blank control replicates prepared at the beginning of the beginning of the exposure period were 55 and 56 mg O2 per dry weight of activated sludge per hour, for the two blank controls prepared after the reference item group were 57 to 54 mg O2/gh and for two replicates prepared at the end of the test were 52 and 49 mg O2/gh. All six values are higher than the 20 mg O2/gh stipulated in the guideline, not more than 30%.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 18 mg/L. The 3-hour EC50 is within the Guideline-recommended range of 2-25 mg/l, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
Test item :
The 3h-EC20, EC50 and EC80 and their 95%-confidence limits could not be calculated because of the absence of a toxic effect.

Table 1 - Composition of the incubation mixtures

Vessel No.

Identification final nominal conc.

(mg/L)

Aliquot of stock solution

 

 

    (mL)*            (mL)**

Water

 

 

(mL)

Synthetic sewage feed

(mL)

Activated sludge

 

(mL)

                     Inoculum control

1

2

0

0

-

-

-

-

234

234

16

16

250

250

                            3,5-DCP

3

4

5

3.2

10

32

-

-

-

3.2

10

32

230.8

224

202

16

16

16

250

250

250

                    Inoculum control

6

7

0

0

-

-

-

-

234

234

16

16

250

250

                          Test item

8

9

10

173

173

173

6.3

6.3

6.3

-

-

-

227.7

227.7

227.7

16

16

16

250

250

250

11

12

13

345

345

345

12.5

12.5

12.5

-

-

-

221.5

221.5

221.5

16

16

16

250

250

250

14

15

16

691

691

691

25

25

25

-

-

-

209

209

209

16

16

16

250

250

250

17

18

19

1381

1381

1381

50

50

50

-

-

-

184

184

184

16

16

16

250

250

250

20

2762

100

-

134

16

250

21

2762

100

-

134

16

250

22

2762

100

-

134

16

250

                    Inoculum control

23

24

0

0

-

-

-

-

234

234

16

16

250

250

* : Stock solution of test item (13810 mg test item/L)

** : Stock solution of 3,5 -DCP (500 mg/L)

Table 2 - Influence of cocoamidopropylhydroxysultaine (test item) and 3 -5 DCP (reference item) on the Oxygen Consumption of Activated Sludge

Vessel

No.

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate

Inhibition

 

 

%

Ph values

Oxygen concentration (mgO2/L)

Respiration rate

(R)

(mg O2/Lh)

Specific respiration rate (Rs) (mg O2/gh)

start*

end*

start*

end*

                    Control

1

2

6

7

23

24

0

0

0

0

0

0

81.94

84.34

85.80

81.60

77.25

73.73

54.63

56.23

57.20

54.40

51.50

49.16

 

7.7

7.8

7.6

7.6

7.6

7.6

8.4

8.4

8.3

8.3

8.3

8.3

8.1

8.0

8.0

8.0

8.0

8.1

8.3

8.4

8.2

8.3

8.0

8.4

Mean

Standard Deviation

Coefficient of Variation

80.78

4.52

5.59%

53.85

3.01

5.59%

 

 

                    Test item                                                                               Mean

8

173

89.20

59.47

-10.4

 

-7.0

7.6

7.6

7.6

8.3

8.3

8.3

8.2

8.2

8.2

8.3

8.3

8.3

9

173

83.70

55.80

-3.6

10

173

86.40

57.60

-7.0

11

345

87.80

58.53

-8.7

 

-16.4

7.5

7.5

7.5

8.3

8.2

8.2

8.0

8.0

8.0

8.0

8.0

8.2

12

345

100.80

67.20

-24.8

13

345

93.36

62.24

-15.6

14

691

130.50

87.00

-61.6

 

-56.7

7.6

7.6

7.6

8.3

8.3

8.3

8.0

8.0

8.0

8.2

8.2

8.1

15

691

137.10

91.40

-69.7

16

691

112.08

74.72

-38.8

17

1381

93.09

62.06

-15.2

 

-12.4

7.6

7.6

7.6

8.3

8.3

8.3

8.1

8.1

8.1

8.3

8.3

8.3

18

1381

95.83

63.89

-18.6

19

1381

83.52

55.68

-3.4

20

2762

95.49

63.66

-18.2

 

1.2

7.6

7.6

7.6

8.3

8.3

8.3

8.1

8.1

8.0

8.4

8.4

8.2

21

2762

67.60

45.07

16.3

22

2762

76.35

50.90

5.5

                Reference item

3

4

5

3.2

10

32

70.40

58.91

19.02

46.93

39.27

12.68

12.8

27.1

76.5

 

7.6

7.6

7.6

8.3

8.4

8.4

8.0

8.1

8.3

8.3

9.1

8.3

* : Start and end of the 3 -hour incubation period

% inhibition : Increased oxygen consumption rate relative to control

Validity criteria fulfilled:
yes
Remarks:
The variation in respiration rates of controls 1 and 2 after 3 hours contact time : less than 15% (+/-2%) ; The 3-hour EC50 of the reference substance is in the accepted range to 5 to 30 mg/l (6.9 mg/l)
Conclusions:
The test item has no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to and including the test concentration of 2762 mg test item/L, corresponding to 1000 mg active ingredient/L.
Executive summary:

The toxicity of the test item (= substance EC 939-455-3 in aqueous commercial product) to freshwater microorganisms was investigated in a GLP-compliant study performed in accordance with OECD Guideline 209 (Activated Sludge, Respiration Inhibition). The results led to the following conlusions:

- the 3h-NOEC for respiration rate inhibition was ≥ 2762 mg test item/L (corresponding to ≥ 1328 mg solid content/L and ≥ 1000 mg active content/L);

- the 3h-EC10 for respiration rate inhibition was > 2762 mg test item/L (corresponding to > 1328 mg solid content/L and > 1000 mg active content/L);

- the 3h-EC50 for respiration rate inhibition was > 2762 mg test item/L (corresponding to > 1328 mg solid content/L and > 1000 mg active content/L).

The reported effect concentrations are based on nominal concentrations.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4).

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX
See "Documentation and scientific justification of the read-across approach" in section 13.2.
Reason / purpose:
read-across source
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 328 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solid content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: read-across from EC 939-455-3
Conclusions:
By analogy with the source substance EC 939-455-3, a 3h-NOEC of 1328 mg solid content/L on freshwater microorganisms is reported for the target substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18(even numbered) acyl) derivs., hydroxides, inner salts (EC 939-457-4).

Description of key information

As the toxicity to aquatic microorganisms of the substance EC 939-457-4 has not been investigated experimentally, a read-across approach was followed to fill in the endpoint. The toxicity to freshwater microorganisms of a structural analogue substance (= source substance EC 939-455-3) was investigated in a GLP-compliant study (Eisner, 2013) performed in accordance with OECD Guideline 209 (Activated Sludge, Respiration Inhibition). The results led to the following conlusions for the substance:

- the 3h-NOEC for respiration rate inhibition was ≥ 1328 mg solid content/L;

- the 3h-EC10 for respiration rate inhibition was > 1328 mg solid content/L;

- the 3h-EC50 for respiration rate inhibition was > 1328 mg solid content/L.

The reported effect concentrations are based on nominal concentrations. By analogy, the same NOEC, EC10 and EC50 values were reported for the target substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18(even numbered) acyl) derivs., hydroxides, inner salts (EC 939-457-4).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 328 mg/L

Additional information

The toxicity to aquatic microorganisms of the structural analogue substance (= source substance EC 939-455-3) was investigated in a GLP-compliant study (Eisner, 2013) performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and was selected as key study for the endpoint of the target substance EC 939-457-4.