Reaction products of sodium glucoheptonate with zinc sulfate and sodium hydroxide

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:

2016/05/24 - 2017/03/22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For main study, three replicates for treatment and six replicates for control group were prepared. 140 mL of test samples from each replicates were drawn and mixed together for each group. 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:

A quantity of 10 mg DABQUEL COMPLEX ZnP was transferred to a 100 mL volumetric flask and volume was made up to the required mark with the algal cultured medium to obtain the nominal concentration 0.1 mg/mL (stock A). Volumes of 320, 640, 960, 1920 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 0.1, 0.2, 0.3, 0.6 and 1.0 mg DABQUEL COMPLEX ZnP/L, respectively.

Test organisms (species):

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

Details on test organisms:

Name of the Organism: Pseudokirchneriella subcapitata
Strain Number: ATCC 22662
Origin and Source of Culture: American Type Culture Collection10801, University of Manassas, Virginia, 20110-2209, USA
Subculture Maintained at: Section - Ecotoxicology Jai Research Foundation, Valvada, Dist. Valsad, Gujarat May 13, 2010 December 04, 2016
Date of Receipt (at JRF): May 13, 2010
Date of Last Subculture: December 04, 2016
Method of Cultivation: static condition
Pre-Culture: The pre-culture was prepared 2 days prior to the commencement of the study by transferring 1 mL from the latest sub culture into a new culture vessel. The pre-culture was incubated under the same conditions as those required for the test and were used when growing exponentially (5E3 - 10E4 cells/mL). The temperature for pre-culture ranged between 21 - 22 °C.

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 23°C

pH:

7.75 - 8.02

Nominal and measured concentrations:

nominal: 0, 0.1, 0.2, 0.3, 0.6 and 1 mg/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 vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH 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(see table 1). The pH of algal culture medium was observed 7.90 and 7.81.

OTHER TEST CONDITIONS
- Light intensity and quality: 6270 - 6400 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.5 - 0.6
- Test concentrations: 0, 0.1, 0.2, 0.3, 0.6 and 1 mg/L
- Range finding study: A preliminary range finding study was conducted with the test concentrations of 0.0 (control), 0.1, 1.0, 10.0, 50.0 and 100.0 mg DABQUEL COMPLEX ZnP/L. Two replicates were run for each concentration of the test item and three replicates for the control.
- Results used to determine the conditions for the definitive study: The percent inhibition of biomass was 0.99, 93.71, 97.56, 99.10 and 100.53 for the test concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 mg DABQUEL COMPLEX ZnP/L, respectively.
The percent inhibition of growth rate was 0.15, 63.09, 72.94, 85.29 and 92.35 for the test concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 mg DABQUEL COMPLEX ZnP/L, respectively

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:

EC10

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: measured concentration >80 % of nominal concentration

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: measured concentration >80 % of nominal concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.89 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: measured concentration >80 % of nominal concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: measured concentration >80 % of nominal concentration

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: measured concentration >80 % of nominal concentration

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: measured concentration >80 % of nominal concentration

Details on results:

The cell concentration in the control cultures was increased by a factor of 99.2 times within the three-day test period. In the treated groups, biomass was decreased with increased concentration of DABQUEL COMPLEX ZnP during the 72 h exposure period.
The coefficient of variation of average specific growth rate during the whole test period (0-3) in replicate control culture 0.65%. The mean coefficient of variation for day 0-1, 1-2, and 2-3 in control culture was 6.32%.
The percent inhibition of biomass was 0.76, 18.39, 33.82, 67.72 and 95.26 for the test concentrations of 0.1, 0.2, 0.3, 0.6 and 1.0 mg DABQUEL COMPLEX ZnP/L, respectively.
The percent inhibition of growth rate was 0.16, 4.38, 8.76, 23.79 and 59.47 for the test concentrations of 0.1, 0.2, 0.3, 0.6 and 1.0 mg DABQUEL COMPLEX ZnP/L, respectively.
The percent inhibition of yield was 0.13, 18.40, 33.12, 67.13 and 94.42 for the test concentrations of 0.1, 0.2, 0.3, 0.6 and 1.0 mg DABQUEL COMPLEX ZnP/L, respectively.

Results with reference substance (positive control):

ErC50 for growth rate inhibition (0 - 72 h) : 3.46 mg Potassium dichromate/L.

Validity criteria fulfilled:

yes

Conclusions:

The key study with DABQUEL ZnP according to the OECD Guideline 201 (Growth Inhibition Study (72 h)) had the following results:
EbC10 for growth rate inhibition (0 - 72 h) : 0.32 mg DABQUEL COMPLEX ZnP/L
ErC50 for growth rate inhibition (0 - 72 h) : 0.89 mg DABQUEL COMPLEX ZnP/L
NOEC for biomass, growth rate and yield : 0.1 mg DABQUEL COMPLEX ZnP/L
LOEC for biomass, growth rate and yield : 0.2 mg DABQUEL COMPLEX ZnP/L

Executive summary:

A GLP compliant key study with DABQUEL ZnP according to the OECD Guideline 201 (Growth Inhibition Study (72 h)) was performed. The key study had the following results:

EbC10 for biomass inhibition (0 - 72 h) : 0.18 m g DABQUEL COMPLEX ZnP/L

EbC10 for growth rate inhibition (0 - 72 h) : 0.32 mg DABQUEL COMPLEX ZnP/L

EyC10 for yield inhibition (0 - 72 h) : 0.21 mg DABQUEL COMPLEX ZnP/L

EbC20 for biomass inhibition (0 - 72 h) : 0.24 mg DABQUEL COMPLEX ZnP/L

ErC20 for growth rate inhibition (0 - 72 h) : 0.46 mg DABQUEL COMPLEX ZnP/L

EyC20 for yield inhibition (0 - 72 h) : 0.27 mg DABQUEL COMPLEX ZnP/L

EbC50 for biomass inhibition (0 - 72 h) : 0.39 mg DABQUEL COMPLEX ZnP/L

ErC50 for growth rate inhibition (0 - 72 h) : 0.89 mg DABQUEL COMPLEX ZnP/L

EyC50 for yield inhibition (0 - 72 h) : 0.42 mg DABQUEL COMPLEX ZnP/L

NOEC for biomass, growth rate and yield : 0.1 mg DABQUEL COMPLEX ZnP/L

LOEC for biomass, growth rate and yield : 0.2 mg DABQUEL COMPLEX ZnP/L

Description of key information

key study

A key study according to the OECD Guideline 201 with the registered substance was performed with the test organism Pseudokirchneriella subcapitata. The study was conducted to determine the inhibitory effect of different concentrations of the registered substance (Zinc Glucoheptonate, ZnGHA) 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) (Kamle, 2017). The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 0.32 (0.27 - 0.38) mg/L. ErC20 = 0.46 (0.40 - 0.52) mg/L and ErC50 = 0.89 (0.76 - 1.04) mg/L (Kamle, 2017). The test item has a total zinc content of 14.2 %, revealing an ErC50 of elemental zinc of 0.126 mg Zn/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.89 mg/L

EC10 or NOEC for freshwater algae:

0.32 mg/L

Additional information

Impact of Environmental conditions on zinc toxicity

There are several studies underlining the fact, that aquatic zinc toxicity significantly depends on environmental factors like dissolved organic carbon (DOC) content (De Schampelaere et al. 2005, Fisher and Frood 1980), pH-value (Wilde et al., 2006, De Schamphelaere 2005), salinity (Eklund, 2005), metal concentration (De Schamphelaere et al. 2005). In the key study an ErC50 (72 h) value of 0.89 mg/L was obtained for P. subcapitata (Kamle, 2017). De Schamphelaere et al.(2005) studied the impact of pH-value, metal concentration and dissolved organic carbon content on the ErC50 (72 h) value of P. subcaptitata. The value obtained in the key study (Kamle, 2017) slightly exceeds the value found by De Schamphelaere et al. (2005) at a pH of 8, a DOC content of 6 mg/L and a Ca concentration of 65.4 mg/L when converting the value for elemental zinc to the target substance ZnGHA (table 1).

pH-value

De Schamphelaere et al. (2005) demonstrated that the pH-value of the test medium has the most significant impact on zinc toxicity. When the pH was reduced to 5.7, the toxicity of zinc was reduced by about 20-fold (De Schamphelaere et al., 2005). The great protective impact of decreasing pH was also shown for the alga Chlorella sp. Here, a pH decrease from 8.0 to 5.5 lead to a 50-fold increase of the ErC50 (48 h) for Chlorella sp. The reduced toxicity with decreasing pH is explained by competition between protons (H+) and metals for binding sites at the algal cell surface, which is part of the biotic ligand model (BLM) theory and confirmed by measurements of extracellular (cell-bound) metal concentrations (Wilde et al., 2006).

Metal concentration

Besides protons, also the metals Na, Mg and Ca compete with zinc for the binding site, and thus decrease zinc-toxicity (De Schamphelaere et al. 2005).

Complexing constituents

Another factor leading to reduced zinc-toxicity is complexation of zinc ions in the medium. A protective impact of DOC-content was found by Fisher and Frood (2008) and De Schamphelaere et al. (2005). Due to inorganic complexation increasing salinity (Eklund, 2005) and water hardness (De Schamphelaere et al. 2005) decreased zinc toxicity

Conclusion

It was shown, that several environmental factors impact the toxicity of zinc to algae leading to a great variation in the EC-values. In general, algae are very sensitive to zinc. When converting the EC50-values found for elemental zinc in literature to ZnGHA, all values, except for measurements at pH <7 and the value for Chlorella sp. at pH 7, are below 1.5 mg ZnGHA/L. The lowest ErC50 value of the regarded studies is between 0.25 – 0.38 mg ZnGHA/L forNitzschia closterium(Fisher and Frood 1980). The highest values for ZnGHA are found at pH 5.5 with 12.94 mg ZnGHA/L for P. subcapitata (De Schamphelaere et al. 2005) and at pH 5.7 with 17.04 mg ZnGHA/L for Chlorella sp. (Wilde 2006) (table 1). Thus, the value of 0.89 mg ZnGHA/L derived in the key study at neutral pH (Kamle, 2017) lies within the range of the values found in publications.  

Table 1: Effect concentration (EC)-values from studies performed with elemental zinc (Zn²⁺) converted to ZnGHA under consideration of the molecuar weight and the purity

Species	Duration of exposure	Dose descriptor	Basis for effect	elemental Zn in source substance [µg/L]	Zn GHA (75 %) [mg/L]	Impact	Reference
Pseudokirchneriella subcapitata	72 h	EC10	growth rate	0.045	0.32		key study, Kamle17
P. subcapitata	72 h	EC50	growth rate	0.126	0.89
P. subcapitata	72 h	NOEC	growth rate	0.014	0.10
P. subcapitata	72 h	LOEC	growth rate	0.028	0.20
P. subcapitata	72 h	EC50	growth rate	2050	12.94	pH 5.7, DOC 6.28 mg/L, Ca 3.45 mg/L	De Schamphelaere 2005
P. subcapitata	72 h	EC50	growth rate	106	0.67	pH 8.01, DOC 5.89 mg/L, Ca 65.4 mg/L
Chlorella sp.	48 h	EC50	growth rate	2700	17.04	pH 5.5	Wilde 2006
Chlorella sp.	48 h	EC50	growth rate	1680	10.60	pH 6
Chlorella sp.	48 h	EC50	growth rate	970	6.12	pH 6.5
Chlorella sp.	48 h	EC50	growth rate	630	3.98	pH 7
Chlorella sp.	48 h	EC50	growth rate	160	1.01	pH 7.5
Chlorella sp.	48 h	EC50	growth rate	52	0.33	pH 8.0
Ceramium tenuicorne	7 d	EC50	length	20 - 33	0.13 – 0.21	7 ‰ salinity	Eklund 2005
Ceramium tenuicorne	7 d	EC50	length	32 - 61	0.20 – 0.39	20 ‰ salinity
P. subcapitata		NOEC/EC10	growth rate	12.6	0.08		ARCHE 2011
Nitzschia closterium	72 h	EC50	growth rate	226	1.43		Johnson 2007
N. closterium	72 h	EC10	growth rate	84	0.53
N. closterium	72 h	LOEC	growth rate	152	0.96
N. closterium	72 h	NOEC	growthrate	97	0.61
N. closterium	72 h	EC50	growth rate	194	1.22	acclimized to 25 µg Cu/L for 168 days
Chlorella sp.	72 h	EC50	growth rate	110	0.69
Chlorella sp.	72 h	EC10	growth rate	28	0.18
Chlorella sp.	72 h	LOEC	growth rate	44	0.28
Chlorella sp.	72 h	NOEC	growth rate	20	0.13
Chlorella sp.	72 h	EC50	growth rate	98	0.62	acclimized to 2 µg Cu/L for 82 days
N. closterium	72 h	EC50	growth rate	40	0.25	Bass strait clone in “clean” Bass strait test water	Fisher and Frood 1980
N. closterium	72 h	EC50	growth rate	60	0.38	Bass strait clone in “dirty” Corio Bay test water
N. closterium	72 h	EC30	growth rate	40 - 60	0.25 – 0.38	Corio Bay clone in “clean” Bass strait test water
N. closterium	72 h	EC50	growth rate	40 - 60	0.25 – 0.38	Corio Bay clone in “dirty” Corio Bay test water