Copper hydroxide nitrate (Cu2(OH)3(NO3))

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from March 13, 2002 to March 25, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

439-590-3

EC Name:

-

Cas Number:

12158-75-7

Molecular formula:

Cu2H3NO6

IUPAC Name:

copper(2+) hydroxide nitrate

Details on test material:

batch n°: 060248
solubility in water: 153 mg/L (20°C, based on dissolved copper), 300 mg/L (20°C, based on test substance)
vapour pressure: < 0.00052 Pa at 25°C
percent purity: > 99%
Cu content (measured): 52.6%
storage conditions: ambient temp, dark, dry

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test:
Five concentrations of Basic Copper Nitrate were used, spaced by a factor of 10 from 0.01 to 100 mg/L. A stock solution of 300 mg/L was prepared and was diluted in steps of 0.1. From each dilution step, 166.7 mL were added to each reaction vessel and were made up to 500 mL with tap water, synthetic wastewater and sludge. Measurements of oxygen uptake of the activated sludge were performed 30 min. and 3 hours after starting aeration.
Main test:
The main test was performed with 6 concentrations of Basic Copper Nitrate from 300 mg to 9.38 mg/L, spaced by a factor of 0.5, including 2 controls and 3 concentrations of the toxic standard dichlorophenol (5, 15 & 30 mg/L). A stock solution of 528.17 mg/L in tap water was prepared and was diluted by steps by a factor of 2. 284 mL of the solutions were transferred into the test vessels to give the calculated find test concentration after addition of synthetic wastewater, tap water and activated sludge.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge from the municipal treatment plant of Pforzheim, Germany was used as a microbial inoculum for the test. This plant predominantly is treating domestic sewage.
2 L of sludge with an initial content of MLSS (mixed liquid suspended solids) of 8 g/L was collected prior one day to the test. It was washed with tap water by centrifugation, resuspended in 4 L of tap water and 200 mL of synthetic wastewater and aerated with an air pump over night.
The MLSS were adjusted to a final concentration of 1.6 g/L in the test medium.
Synthetic water: the test was performed using a synthetic wastewater which was prepared with the following amounts of substances in 1 litre of water:
peptone: 16.0 g
meat extract: 11.0 g
urea: 3.0g
NaCl: 0.7g
CaCl2.2HO: 0.4g
Mg.SO4.7H2O: 0.2g
K2HPO4: 2.8g

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20 +/- 2°C

Nominal and measured concentrations:

nominal concentrations of copper nitrate (mg/L): 9.38 - 18.75 - 37.5 - 75 - 150 - 300

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

130.34 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

copper nitrate

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The respiration rates were calculated from the slopes of the linear part of the recorder trace as mg O2/L/h between approximately 6.5 mg O2/L and 2.5 mg O2/L over a 10 minutes period.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorphenol was in the accepted range 5 to 30 mg/L after 3 hours.

Any other information on results incl. tables

Respiration inhibition in the main test:

30 min.	R	RC1 + RC2	% inhibition*
Copper Nitrate (mg/L)	(mgO2/L/h)	(mgO2/L/h)
0(1)	29,038	61,855	6,11
9,38	31,14	61,855	-0,68
18,75	31,57	61,855	-2,13
37,5	28,60	61,855	7,52
75	23,03	61,855	25,52
150	17,46	61,855	43,55
300	15,88	61,855	48,67
0(2)	32,817	61,855	-6,11
DCP 5	23,16	61,855	25,12
DCP 15	12,28	61,855	60,29
DCP 30	6,33	61,855	79,53

3 hours	R	RC1 + RC2	% inhibition*
Copper Nitrate (mg/L)	(mgO2/L/h)	(mgO2/L/h)
0(1)	27,826	54,480	-2,15
9,38	29,40	54,480	-7,94
18,75	26,21	54,480	3,78
37,5	22,52	54,480	17,35
75	14,91	54,480	45,28
150	11,57	54,480	57,53
300	9,49	54,480	65,16
0(2)	26,654	54,480	2,15
DCP 5	21,94	54,480	19,46
DCP 15	9,83	54,480	63,91
DCP 30	4,98	54,480	81,70

* a negative value means a value > average of controls.

The inhibition data showed toxic effects of Basic Coppe Nitrate from 37.5 to 300 mg/L after 30 min. and from 18.75 to 300 mg/L after 3 hours.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The 2 control respiration rates are within 15% of each other. The EC50 of 3,5-dichlorophenol was in the accepted range 5 to 30 mg/L after 3 hours.

Conclusions:

The effect of Basic Copper Nitrate on a sewage treatment was tested with the activated sludge respiration inhibition test. In a range-finding test which preceeded the main test, effects of the test substance were detected after 3 hours at 100 mg/L. In the main test, which was performed with 6 concentrations between 9.38 and 300 mg/L, spaced by a factor of 2, effects were detected from 18.75 to 300 mg/L after 3 hours. As the effect after 30 min. was < 50% at any test concentration, an EC50 could be calculated only for 3 hours values. It was determined to be 130.34 mg/L test substance (nominal concentration of Copper nitrate in the test solutions).