Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23rd August, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL:
- Source and lot/batch No.of test material: CUR09385 lot 4293484
- Expiration date of the lot/batch: January 2021
- Purity: 97.5%
- Purity test date: 30 June 2021
- Appearance: Clear to cloudy liquid, dark brown-green
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Aliquots of the control and test concentrations were collected from an extra replicate prepared specifically for TOC analysis. This replicate contained only dilution water, synthetic sewage feed, and the test item in appropriate concentrations. 100 mL of each replicate and control was collected.
- Sample storage conditions before analysis: Samples were stored in amber glass bottles and refridgerated at 2-8° C until shipment for analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance directly weighed into the test vessel and distilled water added
- Controls: Two negative controls (DI dilution water only).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: JAMES Wastewater Treatment Plant- Abbotsford BC
- Preparation of inoculum for exposure: Upon arrival at the laboratory, sludge was poured through a 900 µm² Nytex mesh to remove large particles. The sludge was thn split into 500 mL centrifuge bottles and spunat 2500 rpm for 10 minutes (4°C). Supernatant was discarded and the concentrated sludge was resuspended in dechlorinated tap water. The washing step (centrifugation and suspension) was repeated twice more. The washed sludge was then brought up to 14L with dechlorinated tap water, placed in a glass jar, wrapped in aluminum foil, aerated and mixed at test tempearture until use (2 days after collection). Sludge was fed with synthetic sewage feed (50 mL synthetic sewage feed).

The day before the test, aliquots of sludge were dried to determine solids content and was further diluted to 3 g sludge solids/L, resulting in a nominal concentration of 1.5 ± 0.2 g suspended solid/L in test vessels. Samples were taken again at the test initiation showed 3.1 g suspended solids/L, resulting in 1.55 g suspended solids/L in test vessels.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20-20.5 °C
pH:
7.3-8.0
Nominal and measured concentrations:
Nominal: 0 (dilution water control), 62.5, 125, 250, 500, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 2L Glass jars, containing 500 mL volume
- Type: open during aeration
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3 of two contols (total of 6 replicates)
- No. of vessels per abiotic control: None
- Sludge concentration: 3.1 g suspended solids/L
- Nutrients provided for bacteria: synthetic sewage per OECD 209
-Test setup: For the exposure period, flasks recieved either pure test substance or reference substance stock solution. The reference subtance test was done on a different day. Innoculum at a concentrations of ~1.5± 0.2 g solid/L, 16 mL synthetic sewage, and deionized water were added to a test vessel to give a final volume of 500 mL.
- Nitrification inhibitor used: None
- Biomass loading rate: 1.55 g suspended solids/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated city tap water

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Details on termination of incubation: After the 3-hour exposure, a sample was transferred to a BOD bottle. The BOD bottle was then tapped to dislodge bubbles and a self stirring DO probe (YSI Model 58 Dissolved Oxygen Meter) was inserted and probe stirrer turned on. Measurements were taken every 30 seconds for 10 minutes until the DO reading was 0.0 mg/L Data outside the 2-7 minute range was removed.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): DIssolved oxygen concentration. Dissolved oxygen depletion and time interval were trimmed from two to seven minutes, correlated, and the slope of the regression line was calculated as the respiration rate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Range finding study : Yes. A preliminary test noted an effect observed in the 1000 mg/L concentration, but this test was not valid as the reference toxicant did not meet the validity criteria.
- Test concentrations: blank (deioninzed water), 1, 10, 100, 1000 mg/L
- Interval between the substance test and the reference test: 2 days
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
410.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
95% Confidence Interval:369.1 mg/L - 462.2 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Blank controls oxygen uptake rate: 70.2 mg O2/(h.g SS)
- Coefficient of variation of oxygen uptake rate in control replicates: 6%
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- EC50: 13.5 mg/L. The EC50 was within the 5 to 25 mg/L range considered acceptable for the test. There was a two day period between the tests (21st August, 2019).
Reported statistics and error estimates:
NOEC was not determined in study report. The EC10 was not considered reliable due to enhancement of respiration at lower concentrations. NOEC was calculated using a 1-tailed T-test in Microsoft Excel (250 mg/L p=0.26, 500 mg/L p=0.0000001).

Table 1. MTDID 18990 Inhibitory Effect on Respiration

Sample ID

Testing Loading Rate (mg/L)

Aerobic Respiration Rate: R (mg O2/L∙min)

Aerobic Respiration Rate: R (mg O2/L∙h)

Inhibitory Effect on Respiration Rate (%)

Mean Inhibitory Effect on Respiration Rate (%)

Control 1-A

0

1.7400

104.4

-

-

Control 1-B

0

1.6400

98.4

-

-

Control 1-C

0

1.8400

110.4

-

-

Control 2-A

0

1.9200

115.2

-

-

Control 2-B

0

1.8200

109.2

-

-

Control 2-C

0

1.9200

115.2

-

-

Mean Control

-

-

108.8

 -

 -

62.5 mg/L-A

62.5

2.0000

120.0

-10.3

-23.2

62.5 mg/L-B

62.5

2.3000

138.0

-26.6

62.5 mg/L-C

62.5

2.4000

144.0

-32.4

125 mg/L-A

125

2.6000

156.0

-43.4

-30.5

125 mg/L-B

125

2.5000

150.0

-37.9

125 mg/L-C

125

2.0000

120.0

-10.3

250 mg/L-A

250

1.8400

110.4

-1.5

2.9

250 mg/L-B

250

1.8200

109.2

-0.4

250 mg/L-C

250

1.6200

97.2

10.7

500 mg/L-A

500

0.5365

32.2

70.4

68.2

500 mg/L-B

500

0.5879

35.3

67.6

500 mg/L-C

500

0.6079

36.5

66.5

1000 mg/L-A

1000

0.2737

16.4

84.9

83.9

1000 mg/L-B

1000

0.2758

16.5

84.8

1000 mg/L-C

1000

0.3256

19.5

82.0

Table 2. pH adjustment of Test Solutions

Sample ID

Initial pH

Final pH

# of drops of 1N/6N HCL added

Negative Control 1-A

7.4

N/A

-

Negative Control 1-B

7.4

N/A

-

Negative Control 1-C

7.4

N/A

-

62.5 mg/L-A

7.4

N/A

-

62.5 mg/L-B

7.4

N/A

-

62.5 mg/L-C

7.4

N/A

-

125 mg/L-A

7.6

N/A

-

125 mg/L-B

7.6

N/A

-

125 mg/L-C

7.6

N/A

-

250 mg/L-A

7.9

N/A

-

250 mg/L-B

7.9

N/A

-

250 mg/L-C

7.9

N/A

-

500 mg/L-A

8.3

7.9

6 drops, 1N HCl

500 mg/L-B

8.4

7.9

7 drops, 1N HCl

500 mg/L-C

8.4

7.9

7 drops, 1N HCl

1000 mg/L-A

8.9

8.0

3 drops, 6N HCl

1000 mg/L-B

8.9

7.9

3 drops, 6N HCl

1000 mg/L-C

8.9

7.9

3 drops, 6N HCl

Negative Control 2-A

7.3

N/A

 

Negative Control 2-B

7.3

N/A

 

Negative Control 2-C

7.3

N/A

 

N/A: Not applicable, indicates no neutralizing solution was added

Validity criteria fulfilled:
yes
Remarks:
Control oxygen uptake rate > 20 mg O2/gram sludge/hour (70.2 mg O2/(g SS∙h)); coeff. of variation <30% for control replicates (6%); ref. substance EC50 in range 5-25 mg/L (13.5 mg/L)
Conclusions:
3 hour EC50 = 410.3 mg/L in activated sludge for respiration inhibition (OECD 209)
3 hour NOEC = 250 mg/L in activated sludge for respiration inhibition (OECD 209)
Executive summary:

The toxicity of MTDID 18990 to activated sludge was assessed according to the OECD 209 guideline in a definitive test conducted at 62.5, 125, 250, 500, 1000 mg/L. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of MTDID 18990 to activated sludge is 410.3 mg/L. A NOEC of 250 mg/L was calculated via a 1 -tailed T-Test. The study followed an international standard method and is GLP compliant. Therefore the study is deemed reliable without restrictions and can be utilized to calculate a PNEC for STP.

Description of key information

3 hour EC50 of 410.3 mg/L (OECD 209) in activated sludge for respiration inhibition.

3 hour NOEC of 250 mg/L (OECD 209) in activated sludge for respiration inhibition

Key value for chemical safety assessment

EC50 for microorganisms:
410.3 mg/L
EC10 or NOEC for microorganisms:
250 mg/L

Additional information

The toxicity of MTDID 18990 to activated sludge was assessed according to the OECD 209 guideline in a definitive test conducted at 62.5, 125, 250, 500, 1000 mg/L. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of MTDID 18990 to activated sludge is 410.3 mg/L. A NOEC of 250 mg/L was calculated via a 1 -tailed T-Test. The study followed an international standard method and is GLP compliant. Therefore the study is deemed reliable without restrictions and can be utilized to calculate a PNEC for STP.