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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 17th, 2015 to January 14th, 2016.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: samples were taken at the start and at the end of the test.
- Sample storage conditions before analysis: samples were analysed immediately after sampling.
Vehicle:
no
Details on test solutions:
- The solutions containing 100 mg/l, of both the test and a reference item, in the mineral medium, were inoculated.
- Controls: untreated control and positive control were run in parallel.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Source: A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ” Czajka”, Warsaw, receiving predominantly domestic sewage.
- Preparation of inoculum: The coarse particles were removed by settling and the supernatant was discarded. The sludge was washed in the mineral medium. The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated, at the test temperature of 22 °C, until application next day. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
- Amount of inoculum suspended: 30 mg/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
28 d
Test temperature:
22 ± 2°C
pH:
7.02 - 9.20
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: respirometer flasks.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, volume of test solution in flask, V: 0.164 L
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 30 mg/L.
- Nitrification inhibitor used (delete if not applicable): none.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The double-distilled water was taken from redistillation set. It must contain no more than 10% of the organic carbon content introduced by the test material. This was checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents. The measured value was about 3 mg/l of organic carbon.

OTHER TEST CONDITIONS
- Adjustment of pH: no.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Dissolved Organic Carbon, Nitrates, Nitrites.

TEST CONCENTRATIONS
- Justification for using fewer concentrations than requested by guideline: toxicity control at 100 mg/L test item.
Reference substance (positive control):
yes
Remarks:
acetic acid, sodium salt CAS No: 127-09-3, purity ≥ 98.5%, source: Eurochem.
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
If in a toxicity test, containing both the test item and a reference chemical, the biodegradation after 28 days reached 35.4%. Therefore, the test item cannot be considered inhibitory.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- The reference item reached 82.9% of biodegradation after 28 days.

Table 1. Correction for oxygen uptake for interference by nitrification.

                                  days

0

28

difference

 

1) Concentration of nitrate

(mg N-NO3/l)

#13

#14

#15

#13

#14

#15

#13

#14

#15

0.123

0.114

0.161

0.207

0.194

0.211

0.084

0.080

0.050

0.071 ± 0.019

2) Oxygen equivalent (4.57× N-NO3) (mg/l)

 

0.324

 

3) concentration of nitrite

 (mg N-NO2/l)

#13

#14

#15

#13

#14

#15

#13

#14

#15

0.026

0.021

0.018

0.016

0.020

0.015

-0.010

-0.001

-0.003

-0.005 ± 0.005

4) Oxygen equivalent (3.43×N-NO2) (mg/l)

 

-0.017

5) total oxygen equivalent 2) + 4)

 

0.31

Table 2. Sample oxygen uptake: biodegradability.

 

time, days

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/l

a1

3,2

6,2

9,5

13,0

17,9

22,0

24,7

28,4

34,0

38,6

41,4

45,2

a2

6,1

10,9

15,6

19,1

25,9

30,3

34,0

36,9

43,6

47,5

50,7

54,9

a3

4,6

8,2

14,0

18,8

25,8

29,6

32,1

34,5

39,6

43,1

47,2

49,5

am. avg

4,6

8,4

13,0

17,0

23,2

27,3

30,3

33,2

39,1

43,1

46,5

49,9

Blank test O2uptake. mg/l

b1

b2

9.4

14.0

17.6

20.5

22.7

24.9

25.7

27.2

30.0

32.4

33.1

33.7

b3

10.0

12.8

15.3

17.4

19.7

24.3

25.8

26.3

30.9

31.3

33.5

34.2

 . avg

9.7

13.4

16.5

18.9

21.2

24.6

25.8

26.7

30.5

31.8

33.3

34.0

Reference item O2uptake. mg/l

w1

44.9

57.5

67.3

73.8

81.6

86.6

90.7

93.1

95.7

98.5

101.1

103.7

w2

47.2

59.3

68.0

73.9

80.5

84.9

86.4

87.2

90.0

90.3

92.8

96.4

w3

44.4

55.8

63.2

71.0

76.3

81.0

84.9

88.0

92.4

92.8

95.4

95.7

wm. avg

45.5

57.5

66.1

72.9

79.4

84.2

87.4

89.4

92.7

93.9

96.4

98.6

Toxicity control O2uptake. mg/l

a4tox1

48,8

57,9

68,2

75,8

83,3

89,6

95,5

102,1

112,7

117,3

123,4

127,9

a5tox2

46,9

56,8

64,6

70,9

79,6

86,3

92,3

95,7

104,2

107,3

110,0

114,3

a6tox3

49,4

64,2

76,5

85,3

93,2

99,2

104,0

107,0

114,3

118,6

121,2

124,4

toxm. avg

48,4

59,6

69,8

77,4

85,4

91,7

97,3

101,6

110,4

114,4

118,2

122,2

Corrected

test item O2uptake, mg/l

a1- bm

-6,5

-7,3

-6,9

-5,9

-3,4

-2,5

-1,1

1,7

3,5

6,8

8,1

11,2

a2- bm

-3,6

-2,5

-0,9

0,2

4,7

5,8

8,2

10,1

13,2

15,7

17,4

20,9

a3-bm

-5,1

-5,2

-2,5

-0,1

4,5

5,1

6,4

7,7

9,2

11,2

13,9

15,6

Reference item

% degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/l

R1(w1)

45.2

56.5

65.2

70.4

77.4

79.6

83.3

85.0

83.6

85.5

86.8

89.4

R1(w2)

48.1

58.8

66.0

70.6

75.9

77.3

77.8

77.5

76.3

75.0

76.3

80.0

R3(w3)

44.5

54.3

59.9

66.8

70.6

72.3

75.8

78.5

79.5

78.1

79.6

79.1

Rtoxavg

45.9

56.5

63.7

69.2

74.6

76.4

78.9

80.4

79.8

79.5

80.9

82.9

 The result b1is not taken into average value calculations, as it is distinctly greater than other blank test O2uptakes.

Table 3. Toxicity test.

                                  days

0

28

difference

 

1) Concentration of nitrate

(mg N-NO3/l)

#16

#17

#18

#16

#17

#18

#16

#17

#18

0.171

0.152

0.131

0.259

0.241

0.212

0.088

0.089

0.081

0.086 ± 0.004

2) Oxygen equivalent (4.57× N-NO3) (mg/l)

 

0.393

 

3) concentration of nitrite

 (mg N-NO2/l)

#16

#17

#18

#16

#17

#18

#16

#17

#18

0.020

0.031

0.018

0.014

0.018

0.012

-0.006

-0.013

-0.006

-0.008 ± 0.004

4) Oxygen equivalent (3.43×N-NO2) (mg/l)

 

-0.027

5) total oxygen equivalent 2) + 4)

 

0.37

Table 4. pH values of the test flasks (no adjustment of pH was conducted).

flask #

13

14

15

1

2

3

10

11

12

16

17

18

Test item

Control

Reference item

Toxicity test

initial

7.55

7.52

7.55

7.35

7.47

7.48

7.43

7.41

7.30

7.57

7.58

7.47

final

7.55

7.56

8.23

7.35

7.29

7.62

8.30

8.54

8.58

9.20

9.15

9.08

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks'.
Conclusions:
The test item was not found to be inhibitory to microorganism growth.
Executive summary:

A toxicity control was performed during the biodegradability testing of the test item, according to OECD 301F, under GLP conditions. The solutions containing 100 mg/l, of both the test and a reference item, in the mineral medium, were inoculated. The consumption of oxygen was determined from the change in pressure in the apparatus. The carbon dioxide, evolved during test item degradation, was absorbed in a solution of potassium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of calculated ThOD of the test item. Under test conditions, the test item was not found to be inhibitory, reaching a degradation of 35.4% after 28 days.

Description of key information

Key study: Method according to OECD 301F, GLP study. The test item was not inhibitory to microorganisms at a concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

Key study. A toxicity control was performed during the biodegradability testing of the test item, according to OECD 301F, GLP study. Under test conditions, the test item was not found to be inhibitory, reaching a degradation of 35.4% after 28 days.

Supporting studies: The toxicity of the test item on Tetrahymena pyriformis was studied using a method by Schulz (1983). Based on results, the test item has an IC50 of 392.91 (95% CL: 322.01 - 458.42) mg/L. A Microtox(TM) assay was also performed on the test item, according to the method by Kaiser (1987), to assess its toxicity to Photobacterium phosphoreum, after exposure times of 5, 15 and 30 min. The test showed a 30min-EC50 = 284 mg/L on Photobacterium Phosphoreum.

Based on the available information, the lowest EC50 of the test item on microorganisms is ≥ 100 mg/L.

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