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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 May 24th to June 21st, 2016.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
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)
Test material information:
Composition 1
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 A sample was withdrawn just before use for the de termination of the dry weight of the suspended solids.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:
7.09-8.41
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

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:
NOEC
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 54.1% (>25%). Therefore, the test item is not inhibitory.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- The reference item reached 86.6% of biodegradation after 28 days.

Table 1. Sample oxygen uptake: biodegradability.

 

time, days

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/l

a1

64.7

90.7

104.6

116.3

127.8

133.5

137.5

141.5

148.9

152.7

156.4

159.9

a2

65.6

90.5

106.8

116.1

128.4

133.2

138.6

141.7

148.5

151.6

153.9

160.3

a3

69.1

88.7

102.1

114.2

121.6

129.5

132.0

138.5

149.8

152.4

155.6

159.2

am. avg

66.5

90.0

104.5

115.5

125.9

132.0

136.0

140.6

149.1

152.2

155.3

159.8

Blank test O2uptake. mg/l

b1

7.2

12.7

17.4

19.8

22.7

24.7

28.4

29.8

30.7

32.6

36.6

44.4

b2

7.0

13.2

16.5

20.0

23.8

24.8

28.8

30.5

33.4

35.5

38.8

44.5

b3

10.9

13.3

13.9

16.9

17.8

20.7

22.6

24.6

30.0

33.1

39.2

45.0

bm. avg

8.4

13.1

15.9

18.9

21.4

23.4

26.6

28.3

31.4

33.7

38.2

44.6

Reference item O2uptake. mg/l

w1

54.9

64.7

74.3

79.6

83.9

87.9

92.9

96.4

103.2

109.3

111.5

117.2

w2

55.1

66.9

74.6

78.9

86.0

89.6

92.8

95.7

101.3

105.6

110.4

115.4

w3

53.8

66.6

73.8

79.3

83.6

86.3

89.3

92.5

98.5

100.2

104.1

104.0

wm. avg

54.6

66.1

74.2

79.3

84.5

88.0

91.7

94.9

101.0

105.0

108.6

112.2

Toxicity control O2uptake. mg/l

tox1

58.1

81.0

109.5

130.6

141.4

146.9

152.3

155.0

166.9

169.7

172.0

179.7

tox2

58.0

82.4

107.6

132.0

146.1

152.9

158.4

163.0

173.8

179.6

186.7

197.0

tox3

57.9

79.9

104.2

125.4

141.5

146.9

152.6

158.4

166.0

173.4

174.0

185.0

toxm. avg

58.0

81.1

107.1

129.3

143.0

148.9

154.4

158.8

168.9

174.3

177.6

187.3

Corrected

test item O2uptake, mg/l

(a1-bm)

56.4

77.6

88.6

97.4

106.3

110.0

110.9

113.2

117.5

118.9

118.1

115.3

(a2-bm)

57.2

77.5

90.8

97.2

107.0

109.7

112.0

113.4

117.1

117.9

115.7

115.7

(a3-bm)

60.7

75.7

86.2

95.3

100.1

106.0

105.4

110.2

118.5

118.7

117.4

114.6

Reference item

% degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/l

R1(w1)

59.6

66.2

74.9

77.9

80.1

82.7

85.1

87.3

92.2

96.9

93.9

93.0

R1(w2)

60.0

69.0

75.2

76.9

82.8

84.9

84.9

86.4

89.7

92.1

92.5

90.7

R3(w3)

58.3

68.7

74.2

77.4

79.7

80.7

80.4

82.3

86.0

85.2

84.5

76.1

Rwavg

59.3

68.0

74.8

77.4

80.8

82.8

83.4

85.4

89.3

91.4

90.3

86.6

Test item

% degradation

ThOD = 1.54

mgO2/mg

C = 100 mg/l

Rwavg

36.6

50.4

57.6

63.2

69.1

71.5

72.0

73.5

76.3

77.2

76.7

74.9

R1(a1)

37.2

50.3

59.0

63.1

69.5

71.3

72.8

73.7

76.1

76.6

75.1

75.1

R1(a2)

39.4

49.1

56.0

61.9

65.0

68.9

68.4

71.6

76.9

77.1

76.3

74.4

R3(a3)

37.7

49.9

57.5

62.7

67.8

70.5

71.1

72.9

76.4

77.0

76.0

74.8

Toxicity control

% degradation

 

 

Raavg

21.4

29.3

40.3

48.2

51.7

53.2

54.2

54.6

58.4

58.6

57.7

58.2

R1(tox1)

21.4

29.9

39.5

48.7

53.7

55.8

56.8

58.1

61.4

62.9

64.0

65.7

R2(tox2)

21.4

28.8

38.1

45.9

51.8

53.2

54.3

56.1

58.0

60.2

58.5

60.5

R3(tox3)

21.4

29.3

39.3

47.6

52.4

54.1

55.1

56.3

59.3

60.6

60.1

61.5

 

Table 2. The pH values of test flasks (no adjustment of pH was conducted).

flask #

7

8

9

1

2

3

4

5

6

10

11

12

Test item

Inoculum blank

Reference item

Toxicity test

initial

7.27

7.29

7.30

7.28

7.29

7.31

7.29

7.28

7.28

7.29

7.29

7.25

final

7.12

7.10

7.12

7.09

7.15

7.12

8.12

8.33

8.41

7.87

7.63

8.11

 

Validity criteria fulfilled:
yes
Remarks:
difference of replicates <20%, reference item pass level (60%) on day 5, blank oxygen uptake 44.6 mgO2/l in 28 days, pH inside the range 6-8.5,test item oxygen consumption < 60%. Biodegradation (b. on ThOD) 54.1% in toxicity test: not inhibitory.
Conclusions:
The test item was not 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 not inhibitory, reaching a degradation of 54.1% after 14 days.

Description of key information

Key study. Method according to OECD 301F, GLP study. The test item was not inhibitory to microorganisms (54.1% biodegradation in 14d) at a concentration of 100 mg/L. Therefore, the NOEC value for the test item in microorganisms is greater than 100 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC 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, 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 inhibitory, reaching a degradation of 54.1% after 14 days.