Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfonated

Toxicity to microorganisms

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:

6 June 2017 to 7 September 2017

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

Deviations:

no

GLP compliance:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The biological test system was a consortium of microorganisms common to the activated sludge treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic and facultative aerobic bacteria. The test system was chosen because it is representative of a treatment process that may receive the test substance.
- Differential loading: The initial total suspended solids (TSS) concentration of the activated sludge after removal of the supernatant layer measured
approximately 5587 mg/L. The mean measured TSS concentration of the sludge on the day of testing was approximately 3253 mg/L.
- Controls: The control contained 16.0 mL of synthetic sewage, 234 mL of deionized water, and 250 mL of inoculum for a total volume of 500 mL
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 3,5-Dichlorophenol
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Subsequent reference and test substance dosed biotic mixtures were prepared by combining 16.0 mL of synthetic sewage, the appropriate volume of reference substance stock solution (target volume of 3 mL, 15 mL, and 50 mL for 3, 15, and 50 mg/L respectively) or appropriate amount of test substance (target weights of 0.0050 g, 0.0500 g and 0.5000 g for 10,100, and 1000 mg/L respectively) and an appropriate volume of deionized water needed to achieve a volume of 250 mL. After the deionized water was added, 250 mL of inoculum was added to bring the total volume up to 500 mL. The abiotic mixture contained 16.0 mL of synthetic sewage, 0.5002 grams of test substance, and 484 mL of deionized water for a final volume of 500 mL. Finally, a second control was prepared, in the same manner as the first control. All mixtures were aerated for three hours.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: The biological test system was a consortium of microorganisms common to the activated sludge treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic and facultative aerobic bacteria. The test system was chosen because it is representative of a treatment process that may receive the test substance.
- Name and location of sewage treatment plant where inoculum was collected: Easton Wastewater Treatment Facility, Easton, Maryland
- Method of cultivation: The sludge was sieved using a 2 mm screen and allowed to settle for 30 minutes.
- Preparation of inoculum for exposure: The supernatant above the settled solids was removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled sludge were adjusted to a nominal concentration of approximately 3000 mg/L by dilution with deionized water.
- Pretreatment: Fifty mL of synthetic sewage was added to each liter of adjusted sludge per day. The sludge was maintained at an average temperature of 20 +/- 2deg C and continuously aerated overnight. The pH and total suspended solids concentration of the activated sludge were determined on the day of use in the study.
- Initial biomass concentration: The mixtures were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements.


A stock solution of the reference substance, 3,5-dichlorophenol at a nominal concentration of 500 mg a.i./L, was prepared on May 10, 2017 by dissolving 502.4 mg of the reference substance in 10 mL of 1N NaOH and then adding 20 mL of GenPure water. While stirring, 9 mL of 1N H2SO4 was added to reach the point of incipient precipitation. The solution of 3,5-dichlorophenol was then diluted to a final volume of 1 L with GenPure water. The pH of the reference stock solution was 7.59. The reference substance solution was administered to the reference group by volumetric addition. Volumetric addition is the most appropriate route of administration of aqueous solutions. The following amounts were added to
the reference control test chambers:
- Test vessel 1: 3 mg/L concentration in 3 mL using 231.0 mL of deionized water volume.
- Test vessel 2: 15 mg/L concentration in 15 mL using 219.0 mL of deionized water volume.
- Test vessel 3: 50 mg/L concentration in 50 mL using 184.0 mL of deionized water volume.

The biological test system was a consortium of microorganisms common to the activated sludge treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic and facultative aerobic bacteria. The test system was chosen because it is representative of a treatment process that may receive the test substance.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

not reported

Test temperature:

21-22 Celcius

pH:

7.59

Nominal and measured concentrations:

A control was prepared with 16 mL of synthetic sewage, 250 mL of sludge inoculum, and 234 mL deionized water. Test mixtures were prepared with 16 mL of synthetic sewage, 250 mL of sludge inoculum, the appropriate amount of test substance, and the appropriate amount of deionized water to bring the total volume to 500 mL.

Details on test conditions:

TEST SYSTEM
- Test vessel: The mixtures were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements.
- Type (delete if not applicable): open
- Aeration: yes
- No. of vessels per concentration (replicates): triplicate of highest test concentration
- No. of vessels per control (replicates): duplicate
- No. of vessels per abiotic control (replicates): single
- Sludge concentration (weight of dry solids per volume): 16.0 mL of synthetic sewage, the appropriate volume of reference substance stock solution (target volume of 3 mL, 15 mL, and 50 mL for 3, 15, and 50 mg/L respectively)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized Water

OTHER TEST CONDITIONS
- Adjustment of pH: none

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: An inhibitory dose response effect was not observed for the treatment groups.

Details on results:

- Blank controls oxygen uptake rate: The respiration rates in the two controls were 34.13 and 39.35 mg O2/L/hr. Both controls had respiration rates above or equal to 32.53 mg O2/L/hr, which is the low limit based on the validity criteria for mixtures with 1.63 g dry weight of suspended solids per litre.

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: The validity of the test was supported by the results from the
3,5-dichlorophenol reference group, which resulted in an EC50 value of 13.07 mg/L, with 95 percent
confidence limits of 3 mg/L and 50 mg/L. The EC50 for the reference substance was within the 2 to
25 mg/L range considered acceptable for the test.

Reported statistics and error estimates:

A respiration rate was calculated for each test mixture and expressed in mg O2/L/hour. The rate was calculated using DO values between approximately 6.5 mg O2/L and 2.5 mg O2/L, or over a 10 minute period if the DO did not reach approximately 2.5 mg O2/L.
The respiration rate was calculated using the following equation:
Respiration Rate = [(initial DO – final DO)/(final time – initial time)] × 3600 s/hr

Percent inhibition was calculated using the following equation:
Percent Inhibition = (1-(2RS/(RC1 + RC2)) x 100
where:
RS = oxygen consumption rate at a given concentration of the test or reference substance
RC1 = oxygen consumption rate, Control 1
RC2 = oxygen consumption rate, Control 2
When the dose response pattern (percent inhibition versus test substance concentration) allowed for
the calculation of an EC50 value, the data were analyzed using the computer program of C.E. Stephan.
The program was designed to calculate the EC50 value and the 95% confidence interval by probit
analysis, the moving average, or binomial probability with nonlinear interpolation. Confidence limits
(95%) for the EC50 and the EC50 value itself (when applicable) were determined using binomial
probability with nonlinear interpolation only.

Table 1: Summary of Respiration Inhibition

Treatment	Nominal concentration (mg/L)	Respiration Rate (mg O2/L/hr)	Respiration Inhibition (%)
Control 1	0	34.13	NA
Control 2	0	39.35	NA
Positive control 1	3	34.45	6.24
Positive control 2	15	16.39	55.40
Positive control 3	50	4.82	86.88
Test material	10	35.60	3.10
Test material	100	36.84	-0.27
Test material Replicate 1	1000	36.19	1.50
Test material Replicate 2	1000	33.66	8.3
Test material Replicate 3	1000	37.80	-2.89
Test material abiotic treatment	1000	0.5	NA

 

Validity criteria fulfilled:

yes

Conclusions:

An inhibitory dose response effect was not observed for the treatment groups. The EC50 value calculation for the test substance was greater than 1000 mg/L, the highest concentration tested. The abiotic treatment mixture dosed with 1000 mg/L of the test substance had a respiration rate of 0.5 mg O2/L/hr, indicating that there was no significant uptake or release of oxygen resulting from abiotic reactions of the test substance.

Executive summary:

The potential effect of the test substance on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at nominal concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with the test substance at nominal concentrations of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic control was dosed with the test substance at nominal concentration of 1000 mg/L to discriminate between abiotic uptake by the test substance and microbial respiration. After an exposure period of three hours, the respiration rates of the test solutions were measured using an YSI Model 5000 Dissolved Oxygen Meter.

The respiration rates in the two controls were 34.13 and 39.35 mg O2/L/hr. Both controls had respiration rates above or equal to 32.53 mg O2/L/hr, which is the low limit based on the validity criteria for mixtures with 1.63 g dry weight of suspended solids per liter. The coefficient of variation of the two control respiration rates was 10.06%, and was within the 30% limit established for the test. The validity of the test was further supported by the results from the 3, 5-dichlorophenol reference group, which resulted in an EC50 value of 13.07 mg/L, with 95 percent confidence limits of 3 mg/L and 50 mg/L. The EC50 for the reference substance was within the 2 to 25 mg/L range considered acceptable for the test. 

An inhibitory dose response effect was not observed for the treatment groups. The EC50 value calculation for the test substance was greater than 1000 mg/L, the highest concentration tested. The abiotic treatment mixture dosed with 1000 mg/L of the test substance had a respiration rate of 0.5 mg O2/L/hr showing there was no significant uptake or release of oxygen resulting from abiotic reactions of the test substance.

Treatment	Nominal Concentration mg/L	Respiration Rate mg O2/L/hr	Percent Respiration Inhibition
Control 1	0	34.13	NA
Control 2	0	39.35	NA
3,5-dichlorophenol	3	34.45	6.24
3,5-dichlorophenol	15	16.39	55.40
3,5-dichlorophenol	50	4.82	86.88
Test substance	10	35.60	3.10
Test substance	100	36.84	-0.27
Test substance Replicate 1	1000	36.19	1.5
Test substance Replicate 2	1000	33.66	8.39
Test substance Replicate 3	1000	37.80	-2.89
Test substane Abiotic Treatment	1000	0.50	NA

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The potential effect of the test substance on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at nominal concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with the test substance at nominal concentrations of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic control was dosed with the test substance at nominal concentration of 1000 mg/L to discriminate between abiotic uptake by the test substance and microbial respiration. After an exposure period of three hours, the respiration rates of the test solutions were measured using an YSI Model 5000 Dissolved Oxygen Meter.

The respiration rates in the two controls were 34.13 and 39.35 mg O²/L/hr. Both controls had respiration rates above or equal to 32.53 mg O2/L/hr, which is the low limit based on the validity criteria for mixtures with 1.63 g dry weight of suspended solids per liter. The coefficient of variation of the two control respiration rates was 10.06%, and was within the 30% limit established for the test. The validity of the test was further supported by the results from the 3, 5-dichlorophenol reference group, which resulted in an EC₅₀ value of 13.07 mg/L, with 95 percent confidence limits of 3 mg/L and 50 mg/L. The EC₅₀ for the reference substance was within the 2 to 25 mg/L range considered acceptable for the test. 

An inhibitory dose response effect was not observed for the treatment groups. The EC₅₀ value calculation for the test substance was greater than 1000 mg/L, the highest concentration tested. The abiotic treatment mixture dosed with 1000 mg/L of the test substance had a respiration rate of 0.5 mg O²/L/hr showing there was no significant uptake or release of oxygen resulting from abiotic reactions of the test substance.