Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November/December 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

Modification of Test design. Due to the required volume for dissolved copper analyses, additional flasks were included in the test protocol to perform analytical measurements at intermediate times (i.e. 24 and 48 hours).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analyses have been performed every 24 hours for each test concentration and for the control.

Vehicle:

no

Details on test solutions:

Available information has shown that the test item is not soluble in water. A stock solution was prepared in the test medium (nominal concentration: 500 mg/L), in the dark under magnetic stirring, one week before initiating the algal growth inhibition test.
After membrane filtration (nylon membrane filter, 0.45 µm pore size, 90 mm diameter), the stock solution was diluted in the test medium to achieve the required test concentrations. Most of the copper was undissolved and was removed by filtration.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata, green algae belonging to the order of Chlorococcales;
Origin: Liquid culture obtained from the Culture Center for Algae and Protozoa (strain CCAP 278/4)
Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria, U.K.

The stock culture is maintained in liquid medium. It is grown in conical flasks containing Lefebvre-Czarda medium (volume about
100 ml). An amount of the stock culture is transferred once a week into a flask of fresh medium.

Test type:

static

Water media type:

other: OECD medium according to TG 201

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not available

Test temperature:

22.0 – 22.5°C

pH:

Stock Solution: 8.7
Control: 8.2 (T 0), 7.8 (T 72h)
Concentration range (Test solutions): 8.1 - 8.8 (T 0); 7.9 - 8.2 (T 72h)

Dissolved oxygen:

Not available

Salinity:

Not available

Conductivity:

Not available

Nominal and measured concentrations:

31.25 - 62.5 – 125.0 – 250.0 – 500.0 mg/L (initial nominal concentrations)

Details on test conditions:

Exposure conditions applied in the test are summarized below.

Test medium: OECD Growth medium as described in OECD guideline 201
Vessels: 250 mL conical flasks with air permeable stoppers filled with 100 mL of test solutions
Initial cell concentration: 0.93x104 cells/mL taken from an exponentially growing pre-culture
Test concentrations (initial nominal): 31.25 - 62.5 – 125.0 – 250.0 – 500.0 mg/L
Number of replicates:
Algal growth: 6 for control conditions; 3 per concentration; 1 per concentration, without algae (blank control)
Analysis: 1 per concentration for analysis (with algae) for each sampling time
Incubation temperature: 22.0 – 22.5°C
Incubation illumination: 4520 – 5440 lux
Agitation: Orbital shaking 125 rpm
Duration: 72h
Renewal: None
Determination of algal cells: Every 24 hours (particle counter)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

325.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

ErC50 72 h = 325.5 mg/L (299.9 – 348.2) (nominal concentration), corresponding to an exposure to a mixture of 22.25 µg/L dissolved copper and 108.60 mg/L DABT.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

198 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

ErC10 72 h = 198.0 mg/L (141.9 – 192.3) (nominal concentration), corresponding to an exposure to a mixture of 13.81 µg/L dissolved copper and 67.78 mg/L DABT.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

NOEC: 62.5 mg/L (nominal concentration), corresponding to an exposure to a mixture of 5.07 µg/L dissolved copper and 22.54 mg/L DABT.

Details on results:

The analyses performed have shown that dissolved copper concentrations, even if they were low, were in a range that may affect the growth of Pseudokirchneriella subcapitata.

Results with reference substance (positive control):

Sensitivity of the strain of Pseudokirchneriella subcapitata was checked using potassium dichromate.
The test carried out from 07 to 10 November 2016 gave the following result:
ErC50 72 h: 0.82 mg/L (0.76– 0.88 mg/L)
The ErC50 72 hours was calculated using a log-logistic regression analysis with bootstrap estimation of the 95% confidence limits.
This value is included in the warning limits of the laboratory (mean ErC50 +/- 2 SD) on this species corresponding to two standard deviations from the historic geometric mean of the median inhibitory concentration (0.76 – 1.17 mg/L).
This result confirmed the appropriate sensitivity of the algal strain.

Reported statistics and error estimates:

NOEC:
Statistics have been performed using Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming.
Normality and homogeneity of the data have been checked and the significativity of the effect on growth was determined, by performing a one-way ANOVA (α-value = 0.05) followed by the Bonferroni post-hoc test (α-value = 0.05).

ErCx values:
ErC 10 and ErC 50 72 h values have been calculated using log-logistic regression with bootstrap estimation of 95% confidence limits.

Cell density determined with a particle counter during the definitive test.

Test concentrations (initial nominal concentrations) in mg/L	Cell Density x 10^4cell/mL (particle counter)								pH
	T0	Mean	T24h	Mean	T48h	Mean	T72h	Mean	T0	T72h
Control	0.93 0.93 0.93 0.93 0.93 0.93	0.93	2.66 3.83 2.37 3.37 2.54 3.00	2.96	17.25 15.05 10.94 16.83 12.45 11.28	13.97	84.10 70.98 48.28 70.33 47.23 57.66	63.10	8.2	7.8
31.25	0.93 0.93 0.93	0.93	3.54 3.10 2.87	3.17	12.22 11.77 11.45	11.81	68.18 52.32 62.85	61.12	8.1	7.9
62.5	0.93 0.93 0.93	0.93	2.53 2.75 2.27	2.52	14.73 13.42 12.49	13.55	73.09 71.74 51.50	65.44	8.3	7.9
125.0	0.93 0.93 0.93	0.93	2.01 2.03 1.82	1.95	9.30 9.78 7.08	8.72	42.05 39.34 29.42	36.94	8.5	8.1
250.0	0.93 0.93 0.93	0.93	1.83 1.81 1.91	1.85	6.11 6.51 6.43	6.35	20.30 24.09 24.18	22.86	8.7	8.0
500.0	0.93 0.93 0.93	0.93	1.51 1.48 1.44	1.48	1.60 1.64 2.25	1.83	1.47 1.64 1.45	1.52	8.8	8.2

Average specific growth rates and growth rates inhibitions.

Test concentrations (initial nominal concentrations) in mg/L	Average specific growth rate (days^–1) 0-72h	Mean	CV (%)	Growth rate inhibition (%)
Control	1.502 1.445 1.317 1.442 1.309 1.376	1.399	5.5	/
31.25	1.432 1.343 1.404	1.393	3.3	0.4
62.50	1.455 1.449 1.338	1.414	4.7	-1.1
125.0	1.270 1.248 1.151	1.223	5.2	12.6
250.0	1.028 1.085 1.086	1.066	3.1	23.8
500.0	0.153 0.189 0.148	0.163	13.5	88.3

Validity criteria fulfilled:

yes

Remarks:

Biomass of controls increased by a factor of 67.8, corresponding to a mean specific growth rate of 1.4 day-1. Mean CV for section-by-section specific growth rates for control was 15.8%. CV of average specific growth rates in control replicates was 5.5%.

Conclusions:

The analyses performed have shown that dissolved copper concentrations, even if they were low, were in a range that may affect the growth of Pseudokirchneriella subcapitata.
Consequently, NOEC and ECx values have been expressed as follow:
- NOEC: 62.5 mg/L (nominal concentration)
- ErC10 72 h = 198.0 mg/L (141.9 – 192.3) (nominal concentration)
- ErC50 72 h = 325.5 mg/L (299.9 – 348.2) (nominal concentration)

Executive summary:

The unicellular green algae Pseudokirchneriella subcapitatais was cultured for several generations in a defined medium containing a range of dilutions of stock solution of CuDABT. The test concentrations (31.25 - 62.5 – 125.0 – 250.0 – 500.0 mg/L) were prepared by mixing appropriate quantity of stock solution, growth medium and an inoculum of exponentially growing algal cells. The test concentrations were incubated, under continuous illumination, for 72 hours. Algal cell concentrations have been determined every 24 hours.

Inhibition was measured as a reduction in growth rate relative to control cultures grown under identical conditions.

A stock solution was prepared in the test medium (nominal concentration: 500 mg/L), in the dark under magnetic stirring, one week before initiating the algal growth inhibition test. After membrane filtration (nylon membrane filter, 0.45 µm pore size, 90 mm diameter), the stock solution was diluted in the test medium to achieve the required test concentrations.

The analyses performed have shown that dissolved copper concentrations, even if they were low, were in a range that may affect the growth of Pseudokirchneriella subcapitata. It was therefore impossible to identify which compound was responsible for the observed effects.

Consequently, NOEC and ECx values have been expressed as follow:

-      NOEC:62.5 mg/L(nominal concentration)

-      E_rC₁₀72 h = 198.0 mg/L (141.9 – 192.3) (nominal concentration)

-      E_rC₅₀72 h = 325.5 mg/L (299.9 – 348.2) (nominal concentration)

Description of key information

The key values were determined from concentration of CuDABT concentration of:

- NOEC = 62.5 mg/L

- ErC10 - 72 h = 198.0 mg/L

- ErC50 - 72 h = 325.2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

325.5 mg/L

EC10 or NOEC for freshwater algae:

198 mg/L