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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:
supporting study
Study period:
March 3, 2016 - March 31, 2016.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
Toxicity test.
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test).
Version / remarks:
Toxicity test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 of a predominantly domestic sewage
Details on inoculum:
- Source: aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw, receiving predominantly domestic sewage.
- Name and location of sewage treatment plant where inoculum was collected: "Czajka", Warsaw.
- Preparation of inoculum for exposure: 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 22ºC, until application next day. A sample was withdrawn just before use for the de termination of the dry weight of the suspended solids.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:
14 d
Test temperature:
22 ± 2°C
pH:
7.15 - 9.25
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:
14 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 14 days reached 11.5% (<25%). Therefore, the test item can 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.

Correction for oxygen uptake for interference by nitrification

days

0

28

difference

#13

#14

#15

#13

#14

#15

#13

#14

#15

Concentration of nitrate (mg N-NO3/l)

0.083

0.065

0.072

1.16

0.971

0.924

1.08

0.906

0.852

0.946±0.119

Oxygen equivalent (4.57 x N-NO3) mg/l

 

4.32

Concentration of nitrite (mg N-NO2/l)

0.006

0.007

0.008

0.176

0.167

0.185

0.170

0.160

0.177

0.169±0.009

Oxygen equivalent (3.43 x N-NO2) mg/l

 

0.580

Total oxygen equivalent

 

4.90

Toxicity test

days

0

28

14

difference

 

#16

#17

#18

#16

#17

#18

#16

#17

#18

Concentration of nitrate (mg N-NO3/l)

0.039

0.069

0.061

0.93

1.13

0.738

0.891

1.06

0.677

0.976 ± 0.120

0.677

Oxygen equivalent (4.57 x N-NO3) mg/l

 

4.46

3.09

Concentration of nitrite (mg N-NO2/l)

0.005

0.006

0.005

0.075

0.085

0.095

0.070

0.079

0.09

0.075 ± 0.006

0.09

Oxygen equivalent (3.43 x N-NO2) mg/l

 

0.257

0.309

Total oxygen equivalent

 

4.72

3.399

 

Sample oxygen uptake: biodegradability

 

time, days

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/l

a1

7.6

11.1

11.9

11.7

15.0

16.2

16.5

18.3

20.9

21.5

21.8

23.1

a2

5.2

8.0

7.9

9.1

10.0

12.0

11.7

12.4

12.4

13.9

14.2

15.1

a3

7.1

8.4

8.8

8.8

10.0

11.6

11.3

11.3

14.3

13.3

15.2

16.0

am.avg

6.6

9.2

9.6

9.9

11.7

13.3

13.2

14.0

15.9

16.2

17.1

18.1

Blank test O2uptake. mg/l

b1

17.1

20.3

25.6

28.4

32.8

34.5

37.4

38.2

40.2

42.9

43.4

45.0

b2

12.1

16.6

20.7

23.3

27.1

28.1

30.7

31.9

34.2

33.8

35.5

36.6

b3

13.3

16.1

19.7

22.1

24.6

26.9

28.9

30.8

32.6

34.3

36.7

37.6

bmavg

14.2

17.7

22.0

24.6

28.2

29.8

32.3

33.7

35.7

37.0

38.5

39.7

Reference item O2uptake. mg/l

w1

36.5

37.3

42.6

49.3

57.9

61.8

65.6

69.4

73.5

76.0

77.7

82.2

w2

51.2

63.7

73.8

80.2

87.8

90.6

92.9

96.1

99.0

99.3

101.4

101.6

w3

49.1

61.8

70.8

76.5

82.3

87.1

87.2

90.1

93.0

92.8

96.1

95.7

wm.avg

50.1

62.7

72.3

78.3

85.0

88.9

90.0

93.1

96.0

96.1

98.8

98.6

Toxicity control O2uptake. mg/l

a4tox1

61.3

63.8

67.2

69.3

70.3

71.7

72.7

75.4

77.6

78.5

79.5

79.7

a5tox2

62.1

64.8

66.7

68.5

73.0

73.2

75.0

76.0

78.1

78.6

78.9

79.2

a6tox3

60.1

63.6

66.8

66.6

69.7

70.5

0.0

0.0

0.0

0.0

0.0

0.0

toxm.avg

61.7

64.3

66.9

68.9

71.7

72.5

73.8

75.7

77.8

78.6

79.2

79.5

Corrected

test item O2uptake, mg/l

a1- bm

-6.5

-6.6

-10.0

-12.9

-13.2

-13.6

-15.8

-15.4

-14.7

-15.5

-16.7

-16.6

a2- bm

-9.0

-9.7

-14.1

-15.4

-18.1

-17.8

-20.7

-21.2

-23.2

-23.2

-24.3

-24.6

a3- bm

-7.1

-9.2

-13.2

-15.8

-18.2

-18.2

-21.1

-22.3

-21.3

-23.7

-23.3

-23.8

Reference item % degradation(BOD /

ThOD x C) x 100

ThOD = 0.78

mgO2/ mg

C = 100 mg / l

R1(w2)

47.4

59.0

66.4

71.3

76.5

77.9

77.6

80.0

81.1

79.9

80.6

79.3

R3(w3)

44.7

56.6

62.5

66.6

69.4

73.5

70.3

72.4

73.5

71.5

73.8

71.8

Rtoxavg

46.1

57.8

64.5

68.9

72.9

75.7

74.0

76.2

77.3

75.7

77.2

75.5

w1was not taken into average value calculations, as it was distinctly lower than other reference item O2uptakes. 

a6tox3result from flask #18 was not taken into average value calculation, as this flask was withdrawn from test after 14 day incubation to nitrate and nitrite analysis.

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

flask #

19

20

21

7

8

9

10

11

12

22

23

24

Test item

Control

Reference item

Toxicity test

initial

7.55

7.54

7.54

7.48

7.50

7.54

7.57

7.47

7.46

7.54

7.52

7.52

final

7.51

7.73

7.75

7.27

7.19

7.20

8.37

8.43

8.60

7.49

9.25

9.07

Validity criteria fulfilled:
yes
Remarks:
difference of replicates <20%,reference item pass level (60%) on day 5, blank oxygen uptake 39.7 mgO2/l in 28 days, pH inside the range 6-8.5,test item oxygen consumption < 60%. Biodegradation (b. on ThOD) 11.5% in toxicity test: test item inhibitory.
Conclusions:
The test item was found to be inhibitory to microorganism growth at a dose of 100 mg/L.
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 found to be inhibitory, reaching a degradation of 11.5% after 14 days.

Description of key information

Key study. Method according to OECD 209, GLP study. Under test conditions, the EC50 values were not determinable because of the low solubility of the test item; estimated NOEC values were below 1 mg/L. The following values 3h-ECX (mg/L) were determined: EC5 = 85.1 mg/L, EC10 = 113.4 mg/L, EC15 = 133.4 mg/L, based on total inhibition;  EC5 = 148.6 mg/L, EC10 = 327.8 mg/L, EC15 = 805.8 mg/L, based on heterotrophic inhibition; and EC 5 < 1 mg/L, EC10 = 86.6 mg/L, EC15 = 101.8 mg/L, EC20 = 113.4 mg/L, EC30 = 143.3 mg/L, based on nitrification.

Supporting study. Method according to OECD 301F, GLP study. The test item was inhibitory to microorganisms at a concentration of 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 mg/L

Additional information

Key study: A study on the activated sludge respiration inhibition of the test item was carried out according to OECD 209 (GLP study). The respiration rates of samples of activated sludge from a sewage treatment plant treating predominantly domestic sewage (1.5 g/L suspended solids) and fed with synthetic sewage was measured in an enclosed cell containing an oxygen electrode after a contact time (incubation) of 3 hours, at concentrations of 1.0, 10.0, 100.0, 1000.0, 10000 mg/L (nominal). The inhibition of three different oxygen uptakes was determined, total, heterotrophic only and due to nitrification. Blank controls, abiotic controls and positive controls (3,5 -dichlorophenol, tested at 0.01, 0.1, 1.0, 10.0 and 100 mg/L) were run in parallel. All validity criteria were fulfilled. Under test conditions, the EC50 values were not determinable because of the low solubility of the test item; estimated NOEC values were below 1 mg/L. The following values 3h-ECX (mg/L) were determined: EC5 = 85.1 mg/L, EC10 = 113.4 mg/L, EC15 = 133.4 mg/L, based on total inhibition;  EC5 = 148.6 mg/L, EC10 = 327.8 mg/L, EC15 = 805.8 mg/L, based on heterotrophic inhibition; and EC 5 < 1 mg/L, EC10 = 86.6 mg/L, EC15 = 101.8 mg/L, EC20 = 113.4 mg/L, EC30 = 143.3 mg/L, based on nitrification.

Supporting study: 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 found to be inhibitory, reaching a degradation of 11.5% after 14 days.