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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
12 September - 24 October 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD Guideline 209 with deviations: only 4 test concentrations used in the definitive test; EC50 value for the test item was not calculated; incubation time was outside the recommended time of 3 h at two concentrations of reference substance; O2 concentrations were outside the recommended range at some instances
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
only 4 test concentrations in the definitive test; EC50 value for test item was not calculated; incubation time was outside the recommended time of 3 h at two concentrations of reference substance; O2 concentrations were outside the recommended range
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Amounts of test item (5, 9, 16 and 28 mg) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to high shear mixing at approximately 7500 rpm for approximately 5 minutes followed by magnetic stirring for 24 h in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 18, 32 and 56 mg/L (five replicates of each concentration).
- Controls: Positive control (3,5-dichlorophenol) and blank control (containing an equal volume of activated sludge and synthetic medium)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum for exposure: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (17 L) was fed synthetic sewage sludge (850 mL). At time ‘0’ 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5-1 L/minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Two additional control vessels were then prepared prior to the test item vessels being prepared. Finally two further control vessels were prepared.
- Suspended solids concentration = 3.0 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Hardness:
No data
Test temperature:
20 ± 2 °C
pH:
6.5-7.6
Dissolved oxygen:
- After 30 minutes contact time: 2.6-7.6 mg O2/L; after 3 h contact time: 1.6-2.4 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: 10, 18, 32 and 56 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel (material, size, headspace, fill volume): 500 mL conical flask
- Aeration: During the incubation, all vessels were aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5-1 L/minute.
- No. of vessels per concentration (replicates): Five
- No. of vessels per positive control (replicates): Single
- No. of vessels per blank control (replicates): Six

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, pH was adjusted at 7.0-8.0 if necessary.
- Light intensity: Normal laboratory lighting

SYNTEHTIC SEWAGE FEED:
- Composition of synthetic sewage feed in 1 L of deionised water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration rate of activated sludge

MEASURING EQUIPMENTS:
- After 3 h of incubation, an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
- The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- The pH of the test item dispersions were measured at 0 h and prior to measurement of the oxygen consumption rate after 3 h contact time, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
- Range finding study: 10, 100 and 1000 mg/L
- Definitive study: In the range-finding study, no statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L. Based on this information, the test concentrations of 10, 18, 32 and 56 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3.2, 10 and 32 mg/L)
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
See table 6.1.7/1
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Relevant effect levels: EC50 (3 h): 8.5 (6.6-11) mg/L
Reported statistics and error estimates:
- EC50 values and 95 % confidence limits for the reference item were calculated by Litchfield and Wilcoxon (1949) method.
- One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the control and all test concentrations to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999- 2001).

Table 6.1.7/1: Oxygen consumption rates and percentage inhibition values after 3 h contact time in the definitive test

Nominal concentration (mg/L)

Initial O2 Reading (mg O2/L)

Measurement Period

(minutes)

Final O2 reading (mg O2/L)

O2 consumption rates (mg O2/L/h)

% inhibition

0

(control)

R1

3.8

2

2.1

51

-

R2

4

3

1.8

44

-

R3

4.4

3

2.1

46

-

R4

4.8

4

1.8

45

-

R5

5.3

4

2.2

46.5

-

R6

5.3

4

2.2

46.5

-

10

R1

4.4

3

2.1

46

1

R2

4

3

1.7

46

1

R3

4.8

4

1.7

46.5

0

R4

4.8

4

1.6

48

[3]

R5

4.1

3

1.7

48

[3]

18

R1

4.6

3

2.2

48

[3]

R2

4.4

3

2.1

46

1

R3

4.2

3

1.9

46

1

R4

3.8

2

2.2

48

[3]

R5

3.4

2

1.8

48

[3]

32

R1

5.2

4

2.4

42

10

R2

5.1

4

2.2

43.5

6

R3

5.1

4

2.2

43.5

6

R4

4.4

3

2.1

46

1

R5

5.3

4

2.3

45

3

56

R1

6.2

7

2.1

35.14

24

R2

6.2

7

2.3

33.43

28

R3

5.8

6

2.2

36

23

R4

6.6

8

2.1

33.75

27

R5

6.4

7

2.2

36

23

Reference item

3.2

5.3

5

2.4

34.8

25

10

6.2

10

2.5

22.20*

52

32

8.2

10

7.2

6.00**

87

R1 – R6 = Replicates 1 to 6

* 3 .25 h contact time

** 2.75 h contact time

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 3 h NOEC of read across substance zinc monomethacrylate to microorganisms was considered to be 32 mg/L. Similar results can be expected for zinc dimethacrylate.
Executive summary:

A study was conducted to assess the effect of read across substance zinc monomethacrylate on micro-organisms according to OECD guideline 209 in compliance with GLP. Following a preliminary range-finding test, activated sewage sludge was exposed to aqueous dispersions of the test item at nominal concentrations of 10, 18, 32 and 56 mg/L (five replicates of each) for a period of 3 h at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. Six blank controls were included in the test design. A reference substance (3,5-dichlorophenol) was tested as a positive control at concentrations of 3.2, 10 and 32 mg/L. Toxicity to micro-organisms was assessed by measuring the respiration rate. No statistically significantly toxic effects were shown at the test concentrations of 10,18 and 32 mg/L, however statistically significantly toxic effects were shown at the test concentration of 56 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 h exposure was 32 mg/L. The EC50 (3 h) of the reference substance was 8.5 (6.6-11) mg/L. The coefficient of variation of oxygen uptake in the control vessels was 5.2 % and the specific respiration rate of the controls was 31.00 mg O2/g dw/h; EC50 (3 h) of 3,5-dichlorophenol was in the accepted range 5-30 mg/L. Hence, it met the validity criteria for controls and reference material. Under the test conditions, the 3 h NOEC of read across substance zinc monomethacrylate to microorganisms was considered to be 32 mg/L (Roulstone, 2013). Similar results can be expected for zinc dimethacrylate.

Description of key information

Based on the read across substance zinc monomethacrylate study results, zinc methacrylate 3 h NOEC to microorganisms was considered to be 32 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
32 mg/L

Additional information

A study was conducted to assess the effect of read across substance zinc monomethacrylate on micro-organisms according to OECD guideline 209 in compliance with GLP. Following a preliminary range-finding test, activated sewage sludge was exposed to aqueous dispersions of the test item at nominal concentrations of 10, 18, 32 and 56 mg/L (five replicates of each) for a period of 3 h at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. Six blank controls were included in the test design. A reference substance (3,5-dichlorophenol) was tested as a positive control at concentrations of 3.2, 10 and 32 mg/L. Toxicity to micro-organisms was assessed by measuring the respiration rate. No statistically significantly toxic effects were shown at the test concentrations of 10,18 and 32 mg/L, however statistically significantly toxic effects were shown at the test concentration of 56 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 h exposure was 32 mg/L. The EC50 (3 h) of the reference substance was 8.5 (6.6-11) mg/L. The coefficient of variation of oxygen uptake in the control vessels was 5.2 % and the specific respiration rate of the controls was 31.00 mg O2/g dw/h; EC50 (3 h) of 3,5-dichlorophenol was in the accepted range 5-30 mg/L. Hence, it met the validity criteria for controls and reference material. Under the test conditions, the 3 h NOEC of read across substance zinc monomethacrylate to microorganisms was considered to be 32 mg/L (Roulstone, 2013). Similar results can be expected for zinc dimethacrylate.