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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 30, 2016 - March 25, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
According to OECD Guideline 201 the concentration series should preferably cover the range causing 5-75 % inhibition of algal growth rate. The concentration selected in this study covered only a range from 0 to 56.70 % inhibition of growth rate (see attached table 1).
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
140 mL of test samples from each replicate were drawn and mixed together for each concentration. Collected samples were centrifuged at 2000 rpm for 10 minutes in order to remove algal cell. The representative samples were divided into two equal portions. One portion was sent for test concentration analysis at 0 and 72 h and the second portion was stored at -20 ± 5 °C temperature.
Vehicle:
no
Details on test solutions:
Algal culture medium: Culture medium was prepared by adding stock solution I (10 mL), stock solution II (1 mL), stock solution III (1 mL), and stock solution IV (1 mL) and equated up to 1 L in sterilised deionised water (preparation of the stock solutions is described in attached table). The pH of algal culture medium was observed 7.9 and 7.85.
A quantity of 10 mg DABQUEL COMPLEX CuP was transferred to a 100 mL volumetric flask and volume was made up to the required mark with the algal culture medium to obtain the nominal concentration 0.1 mg/mL (stock A). Volumes of 256, 480, 896, 1696 and 3200 µL from the stock A and 2000 µL algal culture were taken and diluted to 320 mL with sterile algal culture medium in respective beakers and after sampling, transferred to conical flasks of 250 mL capacity to obtain the nominal test concentrations of 0.08, 0.15, 0.28, 0.53 and 1 mg DABQUEL COMPLEX CuP /L, respectively.
Six replicates for the control group and three replicates for the treatment groups were used. The experiment was conducted in 250 mL conical flasks.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Manassas, Virginia, 20110-2209, USA
- Method of cultivation: static condition

ACCLIMATION
1 mL from the latest sub culture was transfered into a new culture vessel during exponential growth (5 x 10^3 - 10^4 cells/mL)
- Acclimation period: 2 days
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 - 23 °C
pH:
7.71 - 8.04
Nominal and measured concentrations:
nominal 0.0 (control), 0.08, 0.15, 0.28, 0.53 and 1.00 mg DABQUEL COMPLEX CuP/L (measured >80% of nominal concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile conical flask
- Material, size, headspace, fill volume: 250 mL
- Initial cells density: 6688 cells/mL
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
The cells in the cultured flasks were maintained in suspension by agitating the test flasks continuously at 100 ± 2 rpm, using an orbital shaker during the test period.

GROWTH MEDIUM/ TEST MEDIUM / WATER PARAMETERS
see table below

OTHER TEST CONDITIONS
- Light intensity and quality: 6450 - 6580 lux with a universal UV- free white fluorescent lamp


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: manual counting using a haemocytometer and a microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.9
- Range finding study: A preliminary range finding study was conducted with the test concentrations of 0.0 (control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg DABQUEL COMPLEX CuP/L. Two replicates were used for each concentration of the test item and three replicates for the control. The percent inhibition of biomass was 1.39, 18.02, 90.08, 96.12 and 99.29 for the test concentrations of 0.01, 0.1, 1.0, 10.0 and 100.0 mg DABQUEL COMPLEX CuP/L, respectively.
The percent inhibition of growth rate was 0.00, 2.18, 53.63, 72.09 and 87.35 for the test concentrations of 0.01, 0.1, 1.0, 10.0 and 100.0 mg DABQUEL COMPLEX CuP/L, respectively.
- Test concentrations: nominal 0.0 (control), 0.08, 0.15, 0.28, 0.53 and 1.00 mg DABQUEL COMPLEX CuP/L (measured >80% of nominal concentration)
Reference substance (positive control):
yes
Remarks:
The validity and reliability of the test system (Pseudokirchneriella subcapitata) and incubation conditions were confirmed earlier by conducting a positive control study using Potassium dichromate as the positive control
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.94 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
measured concentration >80 % of initial concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
measured concentration >80 % of initial concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.08 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
measured concentration >80 % of initial concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
measured concentration >80 % of initial concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
measured concentration >80 % of initial concentration
Basis for effect:
growth rate
Details on results:
Validity Criteria of the Study
The coefficient of variation of average specific growth rates during the test period in the replicate control cultures was 0.00%. Thus, the validity criterion was met.
The cell concentration in the control cultures increased exponentially by a factor of 107.2 within the test period. Thus, the validity criterion was met.
The mean coefficients of variation for day 0-1, 1-2, and 2-3 in control culture was 8.78%. Thus, the validity criterion was met.

The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 0.27 (0.22 - 0.33) mg/L, ErC20 = 0.41 (0.36 - 0.48) mg/L and ErC50 = 0.94 (0.78 - 1.14) mg/L.
The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits for the biomass inhibition were EbC10 = 0.15 (0.13 - 0.18) mg/L, EbC20 = 0.20 (0.17 - 0.23) mg/L and EbC50 = 0.35 (0.32 - 0.39) mg/L.
The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 0.16 (0.14 - 0.19) mg/L, EyC20 = 0.21 (0.19 - 0.24) mg/L and EyC50 = 0.36 (0.33 - 0.40) mg/L.
NOEC for biomass, growth rate and yield was 0.08 mg/L.
LOEC for biomass, growth rate and yield was 0.15 mg/L.
The percent inhibition of biomass, growth rate and yield at the different test concentrations is shown in the attached table.
Results with reference substance (positive control):
The EC50 values in the positive control study with Potassium dichromate were EbC50=1.02 (0.88 - 1.18) mg/L (biomass), ErC50=3.46 (2.82 - 4.25) mg/L (growth rate) and EyC50=1.02 (0.88 - 1.18) mg/L (yield).

table 1: Percentage (%) Inhibition of Biomass, Growth Rate, and Yield

Group

Test Concentration (mg/L)

Percentage (%) Inhibition of

Biomass

(EbC) 0 - 72 h

Growth Rate

(ErC) 0 - 72 h

Yield

(EyC) 0 - 72 h

G1

0.0(Control)

-

-

-

G2

0.08

0.83

0.00

0.41

G3

0.15

18.16

4.01

17.30

G4

0.28

32.81

8.47

33.26

G5

0.53

70.63

24.81

69.39

G6

1.00

94.26

56.70

93.79

Validity criteria fulfilled:
yes
Conclusions:
The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 0.27 (0.22 - 0.33) mg/L, ErC20 = 0.41 (0.36 - 0.48) mg/L and ErC50 = 0.94 (0.78 - 1.14) mg/L.
The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits for the biomass inhibition were EbC10 = 0.15 (0.13 - 0.18) mg/L, EbC20 = 0.20 (0.17 - 0.23) mg/L and EbC50 = 0.35 (0.32 - 0.39) mg/L.
The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 0.16 (0.14 - 0.19) mg/L, EyC20 = 0.21 (0.19 - 0.24) mg/L and EyC50 = 0.36 (0.33 - 0.40) mg/L.
NOEC for biomass, growth rate and yield was 0.08 mg/L.
LOEC for biomass, growth rate and yield was 0.15 mg/L.
Executive summary:

The study was conducted to determine the inhibitory effect of different concentrations of the test item on the growth of the freshwater green algal species (Pseudokirchneriella subcapitata) to obtain the EC10, EC20, EC50 [EbCx (the concentration at which x% inhibition of biomass is observed), ErCx (the concentration at which x% inhibition of growth rate is observed), EyCx (the concentration at which x% inhibition of yield is observed)], the No Observed Effect Concentration (NOEC), and the Lowest Observed Effect Concentration (LOEC). The study was conducted in compliance with the OECD Principles of GLP (1998).

The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 0.27 (0.22 - 0.33) mg/L, ErC20 = 0.41 (0.36 - 0.48) mg/L and ErC50 = 0.94 (0.78 - 1.14) mg/L.

The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits for the biomass inhibition were EbC10 = 0.15 (0.13 - 0.18) mg/L, EbC20 = 0.20 (0.17 - 0.23) mg/L and EbC50 = 0.35 (0.32 - 0.39) mg/L.

The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 0.16 (0.14 - 0.19) mg/L, EyC20 = 0.21 (0.19 - 0.24) mg/L and EyC50 = 0.36 (0.33 - 0.40) mg/L.

Description of key information

Results of key study

A key study according to the OECD Guideline 201 with copper glucoheptonate was performed with the test organism Pseudokirchneriella subcapitata. The study was conducted to determine the inhibitory effect of different concentrations of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata to obtain the EC10, EC20, EC50 [EbCx (the concentration at which x% inhibition of biomass is observed), ErCx (the concentration at which x% inhibition of growth rate is observed), EyCx (the concentration at which x% inhibition of yield is observed)], the No Observed Effect Concentration (NOEC), and the Lowest Observed Effect Concentration (LOEC). The study was conducted in compliance with the OECD Principles of Good Laboratory Practice (GLP) (1998).

The 72 h growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 0.27 (0.22 - 0.33) mg/L, ErC20 = 0.41 (0.36 - 0.48) mg/L and ErC50 = 0.94 (0.78 - 1.14) mg/L (Kamle, 2017). Having a total copper content of 13.4 %, the ErC50 of copper in the test item is 0.126 mg Cu/L.

Impact of environmental conditions on copper toxicity

The toxicity of the target substance Copper Glucoheptonate (Cu GHA) is caused by the chelated copper ions (see Read across statement). Thus, effect concentrations for elemental copper can be applied to calculate effect concentrations of Cu GHA taking into account the molecular weight (table 1).

When compared to the converted EC50 (72 h) values for P. subcapitata from Bossuyt and Janssen (2004), the EC50 (72 h) obtained in the key study is significantly higher. However the NOEC found in the study of Bossuyt and Janssen (2004) exceeds the one found in the key study (table 1). This might be due to the fact that the NOEC by Bossuyt and Janssen (2004) was obtained using algae acclimated to copper concentrations of 12 and 35 µg Cu/L. “As the test set-up allowed for a gradual acclimation and induction of the homeostasis processes, which is probably naturally occurring in surface waters, this NOEC may be of high ecological relevance and hence directly comparable to the field situation” (Bossuyt and Janssen, 2004). The copper content of the culture medium used in the key study was 3.74 x 10-3µg/L (2.205 x 10-11mol/L). This lies within the range found as optimal for the growth of P. subcapitata (2.1 x 10-14– 6.1 x 10-10). Nevertheless, the copper content of unpolluted European waters ranges between 0.4 and 16 µg/L (Bossuyt and Janssen 2004). Thus, the copper concentration in the culture medium is unusual low when compared to natural waters. This may lead to an underestimation of the NOEC. It was shown, that P. subcapitata was able to acquire tolerance to copper. Acclimation of algae in Cu2+ concentrations of > 35 µg Cu/L resulted in increased EC50 values in the growth inhibition test (Bossuyt and Janssen 2004). However, a copper concentration of > 35 µg/L is unusual for natural habitats.

Gatidou and Thomaidis (2007) found a EC50 (96 h) of 8.5 mg Cu/L for elemental copper for the saltwater microalga D. tertiolecta in a growth inhibition study equivalent to the OECD Guideline 201 of 8.5 mg. Applying this value on the target substance, this makes an EC-value of 62.2 mg Cu GHA/L for D. tertiolecta (table 1). This increased value when compared to the EC50 (72 h) in the key study might be caused by lower sensitivity of D. tertiolecta compared to P. subcapitata. Another factor might be the higher salinity of the saltwater medium f/2 used in the test by Gatidou and Thomaidis (2007). It was shown that increased salinity has a protective effect on copper toxicity. This is caused by the fact that in higher salinities more cations can form inorganic complexes and thus become unavailable to the organisms (Eklund 2005). Regarding the marine brown macroalga Padina boergesenii, elemental copper was acutely toxic at concentrations higher than 500 µg Cu/L after an exposure time of four days. Converted to Cu GHA this makes a concentration of 2.56 mg Cu GHA/L. Concentrations of 25, 50, 100 µg Cu/L (128.15, 256.3, 512.6 µg Cu GHA/L) decreased the daily growth rate and the photosynthetic efficiency after a prolonged exposure of 21 days (Mamboya et al. 2001).

Table 1: EC, NOEC and LOEC-values from studies performed with elemental copper (Cu2+) converted to Cu GHA, and from the key study the values for CuGHA were converted to elemental copper

Reference

Dose descriptor

Test organism

EC-values for Cu2+[µg/L]

EC-values for Cu GHA [µg/L]

environmental impact

key study

72h EC50

Pseudokirchneriella subcapitata

126

940

no

NOEC

Pseudokirchneriella subcapitata

10.72

80

no

LOEC

Pseudokirchneriella subcapitata

 20.1

150

no

Eklund 2005

7d EC50

Ceramium tenuicorne

1.9

13.9

low salinity

7d EC50

Ceramium tenuicorne

3.8

27.8

low salinity

7d EC50

Ceramium tenuicorne

7.9

57.9

high salinity

7d EC50

Ceramium tenuicorne

13

95.2

high salinity

Bossuyt and Janssen 2004

12wk NOEC

Pseudokirchneriella subcapitata

35

256.3

acclimation

72h EC50

Pseudokirchneriella subcapitata

37

270.9

no

72h EC50

Pseudokirchneriella subcapitata

88

644.4

acclimation in 0.5 µg Cu/L for > 2 weeks

72h EC50

Pseudokirchneriella subcapitata

124

908

acclimation in 100 µg Cu/L for > 2 weeks

Gatidou and Thomaidis 2007

96h EC50

Dunaliella tertiolecta

8500

62244.1

no

 

Conclusion

Except for the study for D. tertiolecta, the read-across studies reveal an EC50 value for Cu GHA of below 1 mg/L. The effect concentrations are influenced by salinity and acclimation. It is noteworthy, that copper concentrations in test and culture media are reduced when compared to copper contents in the field. This may lead to an underestimation of effect concentrations, as algae are able to acclimate to copper, as was shown for P. supcapitata (Bossuyt and Janssen 2004)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.94 mg/L
EC10 or NOEC for freshwater algae:
0.27 mg/L

Additional information

The EC50 and EC10 values for copper Glucoheptonate were obtained in a GLP-compliant key study according to the OECD guideline 201.