Reaction products of sodium glucoheptonate with copper sulfate and ammonium hydroxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016/05/30 - 2017/03/22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

According to the OECD Guideline 202 the concentrations should be arranged in a geometric series with a separation factor preferably not exceeding 2.2. In this study the separation factor between conc. 1 (0.4 mg/L) and and conc. 2 (0.9 mg/L) is 2.25 and between conc. 3 (0.9 mg/L) and conc. 4 (2.1 mg/L) it is 2.3.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid: An amount of 100 mg DABQUEL COMPLEX CuP was transferred in 100 mL volumetric flask and the volume was made up to the mark with reconstituted water to obtain the nominal concentration of 1.0 mg/mL (stock A). Volumes of 100.0, 225.0, 525.0, 1125.0 and 2500.0 μL from the stock A were taken and diluted to 250 mL with reconstituted water in respective glass beakers of 600 mL capacity to obtain the nominal test concentrations of 0.4, 0.9, 2.1, 4.5 and 10.0 mg DABQUEL COMPLEX CuP/L, respectively.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Storage Temperature:Room temperature
Storage Container:Keep container tightly closed in a cool and well ventilated place
Storage Location :Test Item Control Office, JRF

- Solubility and stability of the test substance in the solvent: Test item was dissolved in reconstituted water; stable up to 48 h (>80% of nominal concentration)

Analytical monitoring:

yes

Details on sampling:

100 mL of test samples from each replicates were drawn and mixed together for each group at 0 and 48 h. The samples were divided into two equal portions. One portion (200 mL) was sent for test concentration analysis and the second portion (200 mL) was stored at -20 ± 5 °C temperature till the study completion.

Vehicle:

no

Details on test solutions:

Based on the results of the preliminary range finding study, six nominal concentrations of 0.4, 0.9, 2.1, 4.5 and 10.0 mg DABQUEL COMPLEX CuP/L (Geometric factor 2.2 - 2.3) along with 0.0 (control) were selected for the main study. An amount of 100 mg DABQUEL COMPLEX CuP was transferred in 100 mL volumetric flask and the volume was made up to the mark with reconstituted water to obtain the nominal concentration of 1.0 mg/mL (stock A). Volumes of 100.0, 225.0, 525.0, 1125.0 and 2500.0 μL from the stock A were taken and diluted to 250 mL with reconstituted water in respective glass beakers of 600 mL capacity to obtain the nominal test concentrations of 0.4, 0.9, 2.1, 4.5 and 10.0 mg DABQUEL COMPLEX CuP/L, respectively.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Gravid female Daphnia magna (showing no signs of stress such as presence of males and ephippia, delay in the production of the first brood, discolouration, etc. and having normal behavior) less than 24 h old (first instar nymph) cultured and bred in the Laboratory of Ecotoxicology, Jai Research Foundation
- Source: The master culture of Daphnia magna was originally procured from the MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium
- Feeding during test: none

ACCLIMATION
- Acclimation period: minimum 48 h
- Acclimation conditions (same as test or not): same
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata)

Test type:

static

Water media type:

other: Reconstituted water with a hardness between 140 and 250 mg/L as CaCO3 was prepared by adding CaCl2, NaHCO3, MgSO4 and KCl to distilled water.

Limit test:

no

Total exposure duration:

48 h

Hardness:

190.4 mg/L as CaCO3

Test temperature:

21.0 ± 0.1 °C

pH:

7.45 ± 0.03

Dissolved oxygen:

8.71 ± 0.23 mg/L

Nominal and measured concentrations:

nominal concentrations of 0.4, 0.9, 2.1, 4.5 and 10.0 mg/L
measured active ingredient concentration > 80% of nominal concentration

Details on test conditions:

The whole study was conducted in a specially designed low temperature water bath preset to a water temperature of 20.0 °C (constant within ± 1°C). Uniform temperature in the water chamber of the water bath was obtained by maintaining a slow flow of water by a motor. The beakers containing the test solutions with daphnids were kept in the water bath. The beakers were held in position by special clamps provided within the chamber of the water bath.
A photoperiod of 16 h light and 8 h darkness cycle was maintained with an automatic timer attached to the water bath. Lighting was provided via fluorescent tubes attached on the lid of the low temperature water bath. Reconstituted water was used as the test media. Environmental parameters, namely, temperature (ranged within 20.1 and 20.9 °C), pH (ranged within 7.61 and 7.77) and dissolved oxygen (8.35 and 9.01 mg/L) were measured at 0, 24, and 48 h and hardness was measured at the beginning of the test (at 0 h). Light intensity was measured daily. All water quality parameters were within the specified limits, as per OECD guideline.

TEST SYSTEM
- Test vessel: Glass beaker of 600 mL capacity
- fill volume: 100 ml
- Aeration: The diluent water was aerated, prior to use, for the test so that dissolved oxygen concentration has reached saturation

- Renewal rate of test solution (frequency/flow rate): no renewal
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
(a) Calcium chloride solution
A quantity of 11.76 g CaCl2.2H2O was dissolved in distilled water and the volume was made up to 1 litre.
(b) Magnesium sulphate solution
A quantity of 4.93 g MgSO4.7H2O was dissolved in distilled water and the volume was made up to 1 litre.
(c) Sodium bicarbonate solution
A quantity of 2.59 g NaHCO3 was dissolved in distilled water and the volume was made up to 1 litre.
(d) Potassium chloride solution
A quantity of 0.23 g KCl was dissolved in distilled water and the volume was made up to 1 litre.
From the above stock solutions a volume of 25 mL each of solution (a) to (d) were taken in a volumetric flask and the total volume was made up to 1 litre by adding distilled water. This media was aerated thoroughly for two days and used for conducting the study.

- Intervals of water quality measurement: 48 h

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h darkness cycle
- Light intensity: 1310 - 1320 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility of the daphnids was assessed by gently swirling the test container for 15 seconds and observing their swimming behaviour. Daphnids unable to swim during the agitation of the test container and remaining settled on the water surface or settled at the bottom of the test vessel were recorded as immobile.

RANGE-FINDING STUDY
- Test concentrations: 0.0 (control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg DABQUEL COMPLEX CuP/L
- number of daphnids per concentration: 10
- replicates: no
- Results used to determine the conditions for the definitive study: The percent immobility observed were 0, 0, 0, 50, 100 and 100% at the test concentrations of 0.0 (control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg DABQUEL COMPLEX CuP/L, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: active ingredient concentration > 80% of nominal concentration

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: active ingredient concentration > 80% of nominal concentration

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: active ingredient concentration > 80% of nominal concentration

Details on results:

The percent immobilisations observed at 24 h were 0, 0, 5, 20 and 45 at the test concentrations of 0.4, 0.9, 2.1, 4.5 and 10.0 mg/L, respectively.
The percent immobilisations observed at 48 h were 0, 35, 55, 80 and 100 at the test concentrations of 0.4, 0.9, 2.1, 4.5 and 10.0 mg/L, respectively.
At 24 h, lethargy was observed in G4 (2.1 mg/L) and G5 (4.5 mg/L), lethargy and at bottom of test vessel was observed in G6 (10.0 mg/L). No immobility and behavioural symptoms were observed in G3 (0.9 mg/L) and G2 (0.4 mg/L).
At 48 h, lethargy was observed in G5 (4.5 mg/L), G4 (2.1 mg/L), G3 (0.9 mg/L). No immobility and behavioural symptoms were observed in G2 (0.4 mg/L).
No immobility, signs of disease or stress, e.g., discoloration or unusual behaviour such as trapping at the surface of water were observed in any of the replicates of G1 (control group). The highest tested concentration of DABQUEL COMPLEX CuP causing no immobilisation and the No Observed Effect Concentration (NOEC) was 0.4 mg/L and the lowest tested concentration causing 100% immobilisation within the 48 h test period was 10.0 mg/L. The Lowest Observed Effect Concentration (LOEC) was 0.9 mg/L over the 48 h exposure period of DABQUEL COMPLEX CuP.
The active ingredient concentration of DABQUEL COMPLEX CuP in test media was within acceptable limit (>80% of nominal concentration).

Results with reference substance (positive control):

The percent immobilisations observed at 24 h were 0, 0, 15, 40 and 55 at the test concentrations of 0.09, 0.20, 0.44, 0.96 and 2.11 mg potassium dichromate/L, respectively.
The percent immobilisations observed at 48 h were 0, 15, 40, 60 and 100 at the test concentrations of 0.09, 0.20, 0.44, 0.96 and 2.11 mg potassium dichromate/L, respectively.
At 24 h, in G6 (2.11 mg/L) and G5 (0.96 mg/L) lethargy, floating at surface of water and at bottom of test vessel were observed. In G3 (0.20 mg/L) at bottom of test vessel was observed. No behavioural symptoms were observed in G4 (0.44 mg/L). No immobility and behavioural symptoms were observed in G2 (0.09 mg/L).
At 48 h, complete immobilisation was observed in all replicates of G6 (2.11 mg/L). In G5 (0.96 mg/L) and G3 (0.20 mg/L) lethargy and floating at surface of water were observed. In G4 (0.44 mg/L) lethargy and at bottom of test vessel were observed. No immobility and behavioural symptoms were observed in G2 (0.09 mg/L). No immobility was observed in any of the replicates of G1 (control group).

- Relevant effect levels:
NOEC: 0.09 mg/L (48 h)
LOEC: 0.2 mg/L (48 h)
EC50: 0.67 mg/L (48h), 1.63 mg/L (24 h)

Validity criteria fulfilled:

yes

Remarks:

Immobilisation in control < 10%. Dissolved O2 concentration > 3 mg O2/L in the control and test vessels. < 10% of daphnids ind control show immobilisation and signs of disease or stress.

Conclusions:

CONCLUSION
EC50 (48 h) : 1.6 mg/L
NOEC (48 h) : 0.4 mg/L
LOEC(48 h) : 0.9 mg/L

Executive summary:

This study was performed to assess the toxic effects on Daphnia magna caused by exposure to the test item under static conditions, according to the OECD guideline 202.

Based on the results of the preliminary range finding study, the concentrations selected for the main study were 0.4, 0.9, 2.1, 4.5 and 10.0 mg/L. In the main study, a 48 h static toxicity test procedure was followed, comprising eight groups. Each group consisted of four replicates with 5 daphnids per replicate.

The stability of the test item in test media was analysed during method validation. The test item in test media was stable up to 48 h (>80% of nominal concentration). Test media was analysed for the active ingredient concentration and stability to monitor the concentration and stability of test solution at 0 and 48 h during main study. The active ingredient concentration in test media was within acceptable limit (>80% of nominal concentration).

The highest tested concentration causing no immobilisation and the No Observed Effect Concentration (NOEC) was 0.4 mg/L and the lowest tested concentration causing 100% immobilisation within the 48 h test period was 10.0 mg/L. The Lowest Observed Effect Concentration (LOEC) was 0.9 mg/L over the 48 h exposure period.

The 48 h EC50 of was determined 1.6 mg/L.

Description of key information

Key study

A key study according to the OECD guideline 202 was performed to assess the toxic effects of Copper glucoheptonate on Daphnia magna under static conditions.

Based on the results of the preliminary range finding study, the concentrations selected for the main study were 0.4, 0.9, 2.1, 4.5 and 10.0 mg/L. In the main study, a 48 h static toxicity test procedure was followed, comprising eight groups. Each group consisted of four replicates with 5 daphnids per replicate.

The stability of the test item in test media was analysed during method validation. The test item in test media was stable up to 48 h (>80% of nominal concentration). Test media was analysed for the active ingredient concentration and stability to monitor the concentration and stability of test solution at 0 and 48 h during main study. The active ingredient concentration of Copper glucoheptonate in test media was within acceptable limit (>80% of nominal concentration).

The highest tested concentration of Copper glucoheptonate causing no immobilisation and the No Observed Effect Concentration (NOEC) was 0.4 mg/L and the lowest tested concentration causing 100% immobilisation within the 48 h test period was 10.0 mg/L. The Lowest Observed Effect Concentration (LOEC) was 0.9 mg/L over the 48 h exposure period (Kamle, 2017).

The EC50 (48 h) was 1.6 mg Copper glucoheptonate/L. As the test item has a copper content of 13.4 %, EC50 (48 h) for copper is 0.2144 mg/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). The EC 50 (48 h) values for elemental copper range from 2.5 to 1455 µg Cu/L (table 1). Thus, the EC50 (48 h) value for elemental copper of 214.4 µg Cu/ml is comparably low. This is caused by the fact that standardized laboratory tests do not account for environmental factors influencing toxicity. It was shown that aquatic copper toxicity significantly depends on environmental factors like dissolved organic carbon content (Kramer et al. 2004, De Schampelaere et al. 2004, and 2002), pH-value, Ca-concentration (De Schamphelaere et al. 2002) and Na-concentration (De Schamphelaere et al. 2004). Sodium reduces copper toxicity, amongst others because of its competition with copper ions at the biotic ligand (De Schamphelaere 2007). Organic carbon content has the most significant effect on copper toxicity, due to its copper binding activity (De Schamphelaere et al. 2002 and 2004, Kramer et al., 2004). The overall variation between EC50 (48 h) values for D. magna tested in different organic carbon concentration is between 7.7 and 1455 µg Cu/L (56.4 and 10654.7 µg Cu GHA/L) (table 1). A linear relationship between the content of dissolved organic carbon and 48 h EC50 values of D. magna were found by Kramer et al.(2004) and De Schamphelaere (2002). 

However, Johnston and Brinkman (2008) point out, that laboratory experiments may underestimate lethal concentrations in field, as it was found for mayflies. One reason could be the fact, that in laboratory test the exposure via diet is not accounted.

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 copper glucoheptonate were converted to elemental copper. OC=organic carbon

Reference	Dose descriptor	Test organism	EC-values for Cu^2+[µg/L]	EC/LC-values for Cu GHA [µg/L]	environmental impact
key study	48h EC50	Daphnia magna	214.4	1600	no
	NOEC	Daphnia magna	53.6	400	no
	LOEC	Daphnia magna	120.6	900	no
Kramer 2004	48h EC50	Daphnia magna	7.7	56.4	OC (lowest concentration)
	48h EC50	Daphnia magna	430	3148.8	OC (highest concentration)
De Schamphelaere 2004	48h EC50	Daphnia magna	50.6	370	OC (lowest concentration)
	48h EC50	Daphnia magna	638	4671.4	OC (highest concentration)
	48h EC50	Daphnia magna	34	248.6	OC (lowest concentration)
	48h EC50	Daphnia magna	1086	7952.6	OC (highest concentration)
Calif. Depart. of Pesticide Reg. 2007	48h LC50	Daphnia magna	730	5342.9	no
Brinkman and Johnston 2008	96h LC50	Rhithrogena hageni	137	1002.9	no
	24h LOEC	Rhithrogena hageni	483	3537.1	no
	24h NOEC	Rhithrogena hageni	256	1874.3	no
De Schamphelaere 2007	48h EC50	Daphnia magna	2.5	18.3	Na (lowest concentration)
	48h EC50	Daphnia magna	29.4	215.3	Na (highest concentration)
	48h EC50	Daphnia magna	4.3	31.5	Na (lowest concentration)
	48h EC50	Daphnia magna	25.9	189.7	Na (highest concentration)
De Schamphelaere 2002	48h EC50	Daphnia magna	115	842.1	OC (lowest concentration)
	48h EC50	Daphnia magna	1455	10654.7	OC (highest concentration)

 

Conclusion

There are several factors influencing the copper toxicity in field which are not accounted for in standardized toxicity tests. Sodium and organic carbon concentrations in test media may be much lower than in field. Therefore, copper effect concentrations determined in the laboratory may overestimate the copper toxicity.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.6 mg/L

Additional information

The EC50 value for copper Glucoheptonate of 1.6 mg/L was obtained in a GLP-compliant key study according to the OECD guideline 202.