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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 March 2017 - 14 April 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD 301F. GLP study.
Qualifier:
according to
Guideline:
other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents.
- Sample storage conditions before analysis: samples were analysed immediately after sampling.


Vehicle:
no
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 A sample was withdrawn just before use for the de termination of the dry weight of the suspended solids.
- Pretreatment: Inocula was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge in mineral medium during 1 day at the test temperature of 22 ºC.
- Initial biomass concentration: 30 mg/L SS
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
28 d
Test temperature:
22 ± 2°C
pH:
7.51-8.74
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, each containing 100 mg/L test item and 30 mg/L SS inoculum.
- No. of vessels per control (replicates):
Inoculum blank: 3 replicates, each containing 30 mg/L SS inoculum.
Procedure control: 3 replicates, each containing 100 mg/L reference item (sodium acetate) and 30 mg/L SS inoculum.
Toxicity control: 3 replicates, each containing 100 mg/L test item, 100 mg/L reference item (sodium acetate) and 30 mg/L SS inoculum.
- Sludge concentration (weight of dry solids per volume): 30 mg/L.
- Nitrification inhibitor used (delete if not applicable): none.

TEST MEDIUM / WATER PARAMETERS
- Preparation of test solutions:
The solutions of the test and reference items were prepared, in separate batches, in mineral medium equivalent to a concentration of 100 mg chemical/litre. The requisite volume of solutions of test and reference items, were introduced respectively, into triplicate flasks. 16.4 mg of test item and 16.4 ml of stock solution of the reference item were introduced into each of the triplicate right flasks. Mineral medium only was added to further flasks (for inoculum controls). Potassium hydroxide solution was added to each of the CO2-absorber compartments. The determined content of inoculum at the end of preconditioning was equal to 3.26 mg suspended solids/l. To give a concentration of suspended solids equal to 30 mg/l in each flask, 1.33 ml of preconditioned suspension of inoculum was added into each flask. The flasks were made up to 164 ml volume with prepared mineral medium.

OTHER TEST CONDITIONS
- Adjustment of pH: no.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Biodegradation % based on oxygen consumption.

TEST CONCENTRATIONS
- Test concentrations: 100 mg/L
Reference substance (positive control):
yes
Remarks:
acetic acid, sodium salt CAS No: 127-09-3, purity ≥ 99.7%, source: CHEMPUR, Piekary Śląskie, Polska.
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Readily biodegradability
Details on results:
The NOEC of the test item was determined to be 100 mg/L since the substance degrades well (79.8% of biodegradation at 28 days) and did not inhibit the biodegradation in the toxicity test (61.7% of biodegradation after 14 days) up to 100 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- The reference item reached 72.4 % of biodegradation and the level for ready biodegradability by 5 days (pass level: biodegradation higher than 60% before day 14)

Table 1. 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

2.1

33.8

138.3

179.3

204.7

227.8

240.5

249.1

256.5

266.8

270.8

276.5

281.5

a2

2.1

34.6

139.5

181.7

204.2

225.9

236.3

250.0

257.2

266.7

273.5

278.7

284.0

a3

4.4

31.0

142.0

181.8

206.4

232.2

243.5

252.0

260.3

268.2

275.6

280.1

287.0

am. avg

2.9

33.1

139.9

180.9

205.1

228.6

240.1

250.4

258.0

267.2

273.3

278.5

284.2

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

bm. avg

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

wm. avg

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

tox1

8.6

57.1

99.7

164.2

214.1

244.0

257.1

270.1

281.5

295.3

302.5

308.5

318.8

tox2

5.3

55.2

108.4

170.5

224.6

254.7

269.8

281.7

294.8

309.5

319.2

327.6

338.4

tox3

5.7

53.6

113.0

172.7

216.9

243.5

256.5

270.6

280.9

293.7

300.2

304.5

312.4

toxm.avg

6.5

55.3

107.1

169.1

218.5

247.4

261.1

274.2

285.7

299.5

307.3

313.5

323.2

Corrected

test item O2uptake, mg/l

(a1-bm)

-3.1

21.3

119.0

154.8

176.4

194.6

206.2

211.7

216.5

221.9

224.2

227.1

228.7

(a2-bm)

-3.2

22.1

120.2

157.2

175.9

192.7

202.1

212.6

217.3

221.7

226.9

229.3

231.1

(a3-bm)

-0.8

18.5

122.7

157.3

178.0

199.0

209.3

214.6

220.4

223.3

229.0

230.7

234.1

Reference item % degradation

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

Rwavg

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

Test item% degradation

ThOD = 2.90

mgO2/mg

C = 100 mg/l

R1(a1)

0.0

7.3

41.0

53.4

60.8

67.1

71.1

73.0

74.7

76.5

77.3

78.3

78.8

R2(a2)

0.0

7.6

41.4

54.2

60.6

66.4

69.7

73.3

74.9

76.5

78.2

79.1

79.7

R3(a3)

0.0

6.4

42.3

54.2

61.4

68.6

72.2

74.0

76.0

77.0

79.0

79.6

80.7

Raavg

0.0

7.1

41.6

53.9

61.0

67.4

71.0

73.4

75.2

76.7

78.2

79.0

79.8

Toxicity test

% degradation

 

R1(tox1)

0.9

12.1

21.9

37.9

50.5

57.3

60.6

63.2

65.6

68.0

69.5

70.4

72.3

R2(tox2)

0.0

11.6

24.2

39.7

53.3

60.2

64.0

66.4

69.3

71.9

74.1

75.6

77.6

R3(tox3)

0.1

11.2

25.5

40.3

51.2

57.1

60.4

63.4

65.5

67.6

68.9

69.3

70.5

Rtoxavg

0.3

11.6

23.8

39.3

51.7

58.2

61.7

64.3

66.8

69.2

70.8

71.8

73.5

 

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

flask #

13

14

15

1

2

3

4

5

6

16

17

18

Test item

Inoculum blank

Reference item

Toxicity test

initial

7.58

7.61

7.61

7.59

7.54

7.65

7.61

7.64

7.62

7.51

7.57

7.59

final

7.33

7.96

7.33

7.20

7.19

7.20

8.48

8.58

8.28

8.28

8.22

8.74

At 28 days of test performance:

- The test item attained 79.8% of biodegradation (>60% reached in a 10-day window within the 28-day period of the test)

- The reference item reached 72.4 % of biodegradation and the level for ready biodegradability by 5 days (>60% reached before day 14)

- In the toxicity test the biodegradation was equal to 61.7% in 14 days (>25% in 14 days). Therefore, the test item is not inhibitory.

- The oxygen uptake of the inoculum blank was equal to 52.9 mg/l in 28 days (<60 mg/l in 28 days)

 

Validity criteria fulfilled:
yes
Remarks:
(difference of replicates <20%, reference item pass level (60%) on day 5, blank oxygen uptake 52.9 mgO2/l in 28 days, pH almost inside the range 6-8.5,test item oxygen consumption > 60%. Biodegradation (b. on ThOD) 61.7% in toxicity test: not inhibitory)
Conclusions:
The NOEC (28d) for microorganism toxicity of the test item was determined to be 100 mg/L (based on biodegradation).
Executive summary:

A ready biodegradability manometric respirometry test was perfomed according to OECD Guideline 301 F. The test item was incubated for 28 days at a concentration of 100 mg/L with 30 mg/L SS activated sludge inoculum under aerobic conditions in a mineral medium. Additionally, to check the possible inhibitory effect of the test item, a toxicity test was run in parallel (100 mg/L test item with 100 mg/L reference substance and 30 mg/L SS activated sludge inoculum) at the same conditions. The NOEC (28d) for microorganism toxicity of the test item was determined to be 100 mg/L since the substance degraded well (79.8% of biodegradation at 28 days) and did not inhibit the biodegradation in the toxicity test (61.7% of biodegradation after 14 days) up to 100 mg/L.


Description of key information

Key study. Test method according to OECD 301F, GLP study. The 28 day-NOEC for microorganism toxicity of the test item was determined to be 100 mg/L (based on biodegradation).


Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Key study: A ready biodegradability manometric respirometry test was perfomed on the test item according to OECD Guideline 301F. Under the test conditions, the 28 day-NOEC for microorganism toxicity of the test item was determined to be 100 mg/L since the substance degraded well (79.8% of biodegradation at 28 days) and did not inhibit the biodegradation in the toxicity test (61.7% of biodegradation after 14 days) up to 100 mg/L.