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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:
March 17, 2017 - April 14, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301F 8Ready Biodegradability: Manometric Respiratory Test)
Version / remarks:
Toxicity test
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
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:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- 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:
- Laboratory culture: Aeration tank of Sewage treatment Plant "Czajika", Warsaw, receiving predominantly domestic sludge.
- Name and location of sewage treatment plant where inoculum was collected: "Czajika", 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 until application. A sample was withdrawn just before use for determination of the dry weight of the suspended solids.
- Pretreatment: Inocula was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge in mineral medium at the test temperature of 22 ºC.
- Initial biomass concentration: 30 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
22 ± 2°C
pH:
6.68-8.58
Nominal and measured concentrations:
100 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: respirometer flasks
- Type: closed
- Material, size, headspace, fill volume: glass, volume 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/mL
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The double-distilled water was taken from redistillation set. It mist contain no more than 10% of the organic carbon content introduced by the test material. this was checked by DOC analysis using spectrophotometer Hatch DR 3900 and Hach-Lange reagents. The measured 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:127-09-3, pure p.a min 99.7, CHEMPUR
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:
After correction for the blank inoculum control and nitrification, the biodegradation in toxicity test on the 28th test day is 72.5% (>25%). The nitrification effects on the 28th day are very modest and it was assumed that those effects on the 14th day are the same as the final ones. Therefore, the test item is not inhibitory.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- The reference item reached 72.4% of biodegradation in the 28th day of the test.

Test item

Table 1.1 Correction for oxygen uptake for interference by nitrification

days

0

28

difference

1) Concentration of nitrate (mg N-NO3/l)

Flask #9

Flask #9

Flask #9

1.78

15.6

13.82

2) Oxigen equivalent (4.57 x N-NO3) (mg/L)

 

63.16

3) Concentration of nitrite (mg N-NO2/l)

Flask #9

Flask #9

Flask #9

0.016

0.077

0.061

4) Oxigen equivalent (3.43 x N-NO2) (mg/L)

 

0.209

5) total oxigen equivalent 2) + 4)

 

63.37

Samples no. 7 and 8 were not analysed because of an incident.

Toxicity test

Table 1.2. Correction for oxygen uptake for interference by nitrification

days

0

28

difference

1) Concentration of nitrate (mg N-NO3/l)

Flask #11

Flask #11

 

0.22

12.10

11.88

2) Oxigen equivalent (4.57 x N-NO3) (mg/L)

 

54.29

3) Concentration of nitrite (mg N-NO2/l)

Flask #11

Flask #11

 

0.018

0.041

0.023

4) Oxigen equivalent (3.43 x N-NO2) (mg/L)

 

0.079

5) total oxigen equivalent 2) + 4)

 

54.37

Samples no. 10 and 12 were not analysed because of an incident.

  

Table 2. The pH values of test flasks

Flask #

7

8

9

1

2

3

4

5

6

10

11

12

Test item

Inoculum blank

Reference item

Toxicity test

Initial

7.20

7.21

7.21

7.59

7.54

7.65

7.61

7.64

7.62

7.12

7.21

7.21

final

6.96

7.00

6.68

7.20

7.19

7.20

8.48

8.58

8.28

7.14

*

7.61

* missed measurement by mistake. No adjustment of pH was conducted

Table 3. Sample oxygen uptake: biodegradability

 

Time, days

1

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/L

a1

1.7

41.6

96.2

130.5

143.8

158.8

163.8

169.1

173.4

182.3

186.6

190.9

197.0

a2

0.8

30.5

74.6

117.3

140.2

152.5

158.6

163.8

168.9

174.2

180.8

183.9

189.6

a3

0.0

29.9

77.4

126.6

138.0

151.1

154.8

158.6

161.6

169.4

174.2

177.8

182.2

amavg

0.9

34.0

82.7

124.8

140.7

154.2

159.1

163.8

168.0

175.3

180.5

184.2

189.6

Blank test O2uptake, mg/L

b1

4.9

10.7

19.6

27.5

31.8

38.0

38.8

43.5

46.5

50.3

52.4

55.0

57.9

b2

4.0

12.0

18.8

22.7

26.2

32.3

34.0

37.5

40.7

46.2

48.9

52.1

56.3

b3

6.9

14.9

19.5

23.2

27.1

29.3

29.9

31.4

32.5

38.2

38.6

41.1

44.4

bmavg

5.3

12.5

19.3

24.5

28.4

33.2

34.2

37.4

39.9

44.9

46.6

49.4

52.9

Reference item O2uptake, mg/L

w1

13.7

50.4

64.5

72.9

77.3

84.5

86.7

88.1

90.8

94.6

96.6

97.2

98.7

w2

15.0

52.8

67.3

76.5

82.1

89.8

93.1

95.7

98.7

103.0

105.8

109.0

111.4

w3

13.2

52.4

67.6

79.0

83.3

90.0

93.1

98.0

100.3

106.7

110.3

113.1

118.0

wmavg

14.0

51.9

66.5

76.1

80.9

88.1

91.0

93.9

96.6

101.4

104.2

106.4

109.3

Toxicity control O2uptake, mg/L

a4tox1

10.3

82.5

139.2

175.8

189.8

202.9

205.0

208.6

213.1

219.5

222.9

225.3

230.2

a5tox2

13.0

80.2

119.4

161.8

190.1

205.4

211.4

217.6

223.0

234.3

238.2

242.8

250.0

a5tox3

9.0

74.3

120.5

167.6

194.9

207.5

217.4

Nitrification measurement on 14thday

toxmavg

10.8

79.0

126.4

168.4

191.6

205.3

211.3

213.1

218.0

226.9

230.6

234.0

240.1

Corrected test item O2uptake, mg/L

a1- bm

-3.5

29.1

76.9

106.0

115.5

125.6

129.5

131.7

133.5

137.4

139.9

141.5

144.1

a2- bm

-4.4

18.0

55.3

92.8

111.8

119.3

124.4

126.3

128.9

129.3

134.2

134.5

136.8

a3- bm

-5.3

17.4

58.1

102.1

109.7

117.9

120.6

121.1

121.7

124.4

127.6

128.4

129.3

Reference item % biodegradation (BOD/ThODxC)x100

 

ThOD=0.78

mgO2/mg

C=100 mg/L

R1(w1)

10.8

48.5

57.9

62.0

62.8

65.8

67.2

64.9

65.3

63.6

64.0

61.2

58.7

R2(w2)

12.4

51.6

61.5

66.6

68.9

72.6

75.5

74.6

75.4

74.5

75.9

76.4

75.0

R3(w3)

10.2

51.1

62.0

69.8

70.4

72.8

75.5

77.6

77.4

79.2

81.6

81.7

83.5

Rtoxavg

11.2

50.4

60.5

66.2

67.4

70.4

72.8

72.4

72.7

72.4

73.9

73.1

72.4

 

Validity criteria fulfilled:
yes
Remarks:
(difference of replicates <20%,reference item reached pass level (60%) on day 5, biodegradation of toxicity test on day 28 was 72.5%,oxigen uptake of blank was 52.9 mg O2/l in 28 days,pH values were inside 6-8.5 range,oxigen consumptiom by test item <60%)
Conclusions:
The test item was not inhibitory to microoganism growth at a dose of 100 mg/L.
Executive summary:

A toxicity control was performed during the biodegradability test of the test item, according to OECD 301F, following GLP. 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 soluion 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 72.5% after 14 days.

Description of key information

Key study: OECD 301F and EU method C.4-D. GLP study. The test item was not inhibitory to microorganism growth at a dose of 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Key study: A toxicity control was performed during the biodegradability test of the test item, according to OECD 301F, following GLP. 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 72.5% after 14 days.