Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Lack of analytical determination of the substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is included in the report (date of inspection: from 22 to 24 January 2013)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solutions of CuGUN have been prepared in deionised water acidified with nitric acid to reach pH < 4 in order to stabilized the test item in solution until use. Indeed, according to information provided by the sponsor, CuGUN is rapidly hydrolyzed in N-guanylurea and copper, at pH 7.
Stock solutions were carried out, for each test and used for carrying out the test concentrations.
Stock solution for definitive test (1 g/L): pH: 6.8; conductivity: 567 µS/cm

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The inoculum was prepared from activated sludge sampled in the aeration tank treatment plant receiving predominantly domestic sewage (Montataire - (60) France).
After settling, the supernatant of activated sludge was discarded and the sludge re-suspended in chlorine-free tap water. The wash-water was again removed after settling of sludge and activated sludge were re-suspended in chlorine-free tap water.
The inoculum was continuously aerated and 50 mL of synthetic sewage feed/L activated sludge were added each day until use.
Sampling of the 10th December 2013 and test carried out 11 December 2013: dry matter of inoculum: 3300 mg/L; pH: 7.7; 227.3 mL of inoculum we added per test flask corresponding to a concentration of suspended solids (SS) in the test mixture of 1.5 g/L.

Test type:

static

Total exposure duration:

3 h

Post exposure observation period:

/

Hardness:

/

Test temperature:

Between 19.5 and 21.0°C

pH:

Between 7.0 and 8.0

Salinity:

/

Nominal and measured concentrations:

Test nominal concentrations: 5.7; 14.8; 38.5; 100 and 260 mg/L

Details on test conditions:

Vessel: 1 litre glass beakers
Dilution water: deionised water
Medium: synthetic sewage feed
Total volume of mixtures: 500 mL
Air supply: clean, oil-free. Air flow 0.5 to 1 litre/ min.
Definitive test: Blank controls: 6 test flasks and test concentrations: 4 test flasks for each

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 73.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 23.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 14.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Details on results:

Statistical tests (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming) were performed to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) in the test conditions.
After verification of the normality and homogeneity of variance,Bonferroni test was performed and showed a statistically significant effect from the test concentration of 38.5mg/L.
Therefore, the first test concentration without effect observed is the test concentration 14.8 mg/L of test item.

ECx(3 hours) were calculated using log-logistic regression with bootstrap estimation of the 95% confidence limit:
EC20 3h = 23.4 mg/L (15.5 – 33.9 mg/L)
EC50 3h = 73.8 mg/L (60.6 – 91.3 mg/L)
EC80 3h: Not determinable

Results with reference substance (positive control):

The EC50 3,5-DCP, after a contact time of 3 hours, was graphically determined between 6.0 and 8.0 mg/L.

Range-finding test: CuGUN

Identification	Q1 (mg/L)	Q2 (mg/L)	Dt	R mg O2/Lh	Rs mg O2/gh	IT (%)	pH
							T0	3h
Blank control 1	6.9	4.2	6	27.0	18.0	/	7.3	7.8
Blank control 2	6.8	4.2	6	26.0	17.3		7.3	7.7
Blank control 3	6.5	3.5	6	30.0	20.0		7.3	7.7
Blank controls	Mean			27.7	18.4	/
CuGUN 1.0 mg/L	7.0	4.2	6	28.0	18.7	- 1.2	7.3	7.8
CuGUN 10.0 mg/L	7.2	4.7	6	25.0	16.7	9.6	7.2	7.8
CuGUN 100 mg/L	8.3	7.1	6	12.0	8.0	56.6	7.0*	7.9
CuGUN 1000 mg/L	9.3	9.2	6	1.0	0.7	96.4	7.0*	7.3
CuGUN Abio. 1000 mg/L (1)	9.2	9.2	6	0.0	0.0	/	7.0*	7.2
CuGUN Abio. 1000 mg/L (2)	9.2	9.2	6	0.0	0.0		7.1*	7.2
CuGUN Abio. 1000 mg/L (3)	9.3	9.3	6	0.0	0.0		7.0*	7.1

Specific inhibitor of nitrification: N-allylthiourea (ATU)

 

Identification	Q1 (mg/L)	Q2 (mg/L)	Dt	R mg O2/Lh	Rs mg O2/gh	IT (%)	pH
							T0	T3h
Blank control 1	7.4	5.3	6	21.0	14.0	/	7.3	8.0
Blank control 2	7.3	5.1	6	22.0	14.7		7.3	8.0
Blank controls	Mean			21.5	14.35	/
CuGUN 1.0 mg/L	7.0	4.6	6	24.0	16.0	- 11.6	7.3	8.0
CuGUN 10.0 mg/L	6.8	4.3	6	25.0	16.7	-16.3	7.3	8.0
CuGUN 100 mg/L	8.0	7.1	6	9.0	6.0	58.1	7.2*	7.9
CuGUN 1000 mg/L	8.7	8.5	6	2.0	1.3	90.7	7.0*	7.2

*:pH adjustment with NaOH was carried out before addition of the inoculum

Validity criteria fulfilled:

yes

Remarks:

The blank controls oxygen uptake rate remained above 20 mg O2/g h during the test and the coefficient of variation did not exceed 30% at the end of the test. EC50 (3 hours) of 3,5-dichlorophenol were in the range 2 mg/L and 25 mg/L

Conclusions:

In the test conditions, an EC20 of 23.4 mg/L, an EC50 of 73.8 mg/L and a NOEC of 14.8 mg/L have been determined for the test item CuGUN, after a contact time of 3 hours.

Executive summary:

The total respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed is measured after a contact time of 3 hours. The respiration rate of the same activated sludge in the presence of various concentrations of the test item: CuGUN under otherwise identical conditions is also measured. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean respiration rates of the controls.

In the test conditions, no oxygen consumption was observed for abiotic controls during this range-finding test, showing a lack of reducing properties of the test item and an EC₂₀ of 23.4 mg/L, an EC₅₀ of 73.8 mg/L and a NOEC of 14.8 mg/L have been determined for the test item CuGUN, after a contact time of 3 hours.

.

Description of key information

The total respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed is measured after a contact time of 3 hours.
The respiration rate of the same activated sludge in the presence of various concentrations of the test item: "CuGUN" under otherwise identical conditions is   also measured.
The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean respiration rates of the controls.
An EC50 value is calculated and a no observed effect concentration (NOEC) is statistically determined from the inhibitory effect observed at different                concentrations when possible.

Key value for chemical safety assessment

EC50 for microorganisms:

73.8 mg/L

EC10 or NOEC for microorganisms:

14.8 mg/L

Additional information

No oxygen consumption was observed for abiotic controls during the range-finding test, showing a lack of reducing properties of the test item.

Statistical tests (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming) were performed to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) in the test conditions. After verification of the normality and homogeneity of variance,Bonferroni test was performed and showed a statistically significant effect from the test concentration of 38.5mg/L.

Therefore, the first test concentration without effect observed is the test concentration 14.8 mg/L of test item.

ECx(3 hours) were calculated using log-logistic regression with bootstrap estimation of the 95% confidence limit:

EC20 3h = 23.4 mg/L (15.5 – 33.9 mg/L)

EC50 3h = 73.8 mg/L (60.6 – 91.3 mg/L)

EC80 3h: Not determinable