Calcium diformate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Scientifically accepted method, but EPI Suite not validated for organic salts. This QSAR program does not not fully meet the OECD Principles for (Q)SAR Validation (inter alia no internal cross-validation and no external validation was performed); for some classes rather small training sets were used as experimental basis; results can nevertheless be used as rough estimation

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Principles of method if other than guideline:

ECOSAR (v0.99h)

Test organisms (species):

other: green algae

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

584 g/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: not specified

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Test performed from 08 Nov 1994 to 11 Nov 1994.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Guideline study under GLP conditions, but test item concentrations not verified analytically and purity of test substance not known

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Strain: 1077/5
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa
- Age of inoculum (at test initiation): from exponentially  growing pre-culture, set up three to four days prior to test start
- Method of cultivation: Algae culture maintained at 20±1 °C under continuous white light in the range 6000 to 10000 lux. The culture was continuously shaken.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 ± 1 °C

pH:

test start: 8.0
test end: 9.0

Nominal and measured concentrations:

nominal: 0 (control), 56, 100, 320, 560, and 1000 mg/L (test material)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250-mL concial flasks; 200 mL test volume
- Aeration: none, but incubation on orbital shaker for 72 h
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 1.0E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes (nutrient  stock solutions according to ISO standards and recommendations of VKI  were then added and the pH adjusted to 8.0±0.2)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater, filtered, sterilized (120 °C, 30 min), aerated (2 h)


OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: white light with 6000 - 10000 lx
- Salinity (for marine algae):


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell density after 0, 24, 48, and 72 h (measured using a  microscope and a haemocytometer)
- Other: pH at test start and test end
- endpoints: inhibition of biomass (EbC50; area under the curve) after 72  h and growth rate (ErC50) between 0 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(biomass integral)

Details on results:

NOEC not stated

Cell density

Concentration (mg/L)	Cell density (cells/mL * 10^4)
	0 h	24 h	48 h	72 h
0	1.0	2.8	33.6	95.1
56	1.0	3.4	30.9	92.3
100	1.0	3.7	42.1	70.2
320	1.0	2.7	36.4	92.7
560	1.0	3.7	39.6	86.4
1000	1.0	4.2	27.0	73.6

 

 

Growth inhibition

Concentration	Biomass		Growth rate
(mg/L)	% inhibition		% inhibition
	48h	72h	48h	72h
56	4	4	2	1
100	-28	4	-6	7
320	-7	-2	-2	1
560	-21	-3	-5	2
1000	10	20	6	6

 

Conclusions:

Although the purity of the test material is not known, it is considered that the study is able to sufficiently demonstrate that marine algae are not more sensitive to formate than freshwater algae.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 10, 1990 to Jan 14, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Procedure for the static algal assay as described in ABC Protocol  #8004-PMN, following methods formulated by:
 
1) Miller et al. (1978). The Selenastrum capricornutum Printz Algal Assay Bottle Test. Experimental Design, Application and Data Interpretation Research Laboratory, Corvallis, Oregon 97330. EPA-600/9-78-018.
2) OTS Algal Acute Toxicity Test, August, 1982. EG-8, ES-5.
3) ASTM (1983). Proposed Standard Practice for Conducting Short-Term Toxicity Tests with Freshwater and Saltwater Algae. E-47.01, Draft No. 5.

GLP compliance:

yes

Remarks:

according to Quality Assurance Officer

Analytical monitoring:

yes

Details on sampling:

- Sampling method: 100 mL was collected at test start and after 96 h and in duplicate from control and each concentration.
- Sample storage conditions before analysis: samples were frozen and shipped to Hoechst Celanese Corp.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ABC Laboratories Inc. culture #W
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas, Austin
- Age of inoculum (at test initiation): Algae cultures for the toxicity test were 5 days old at test initiation.
- Method of cultivation: Algae were culivated in tubes containing ca. 10 mL sterile synthetic algae medium. Flasks were 
incubated and agitated (100 rpm) 


ACCLIMATION
- Culturing media and conditions (same as test or not): 100 mL of sterile synthetic algae medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 °C

pH:

6.9 - 7.4 at test start to 7.8 - 8.1 at test end (96 h); (no concentration dependence)

Nominal and measured concentrations:

nominal: 0 (control), 63,  125, 250, 500, and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000-mL test flasks  
- Type (delete if not applicable): Flasks were closed with a foam plug
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: actual mean contol cell density of 8.5 x  10^3 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes; Sterile algae nutrient medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: To 7.5 +- 1 using 1N NaOH
- Photoperiod: continuous
- Light intensity and quality:  4167-4500 lx ("cool white")

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Cell counts were conducted daily for each test replicate using a hemacytometer and compound microscope.
- Concentration of test material in the media was determined at the beginning and end of the incubation period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 20, 40, 80, 160, and 320 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Algal cells counts in this study were 110, 110, 110, 110 and 86% of the control  population, respectively

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistical re-evaluation

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

807 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistical re-evaluation

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistical re-evaluation

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

63 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: original value

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

570 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: original value

Details on results:

- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

Original evaluation:
Cell counts for each replicate and controls  were subjected to analysis of variance (ANOVA) followed by Dunnett's test accepting p < 0.05 
as significant.
EC50 values were calculated from a regression plot. 
Two regression plots were constructed using either the mean cell count or the log of the cell count. 
The regression equation giving the best fit was used to determine the EC50.
Re-evaluation: The raw data for cell count was used to re-evaluate using ToxRat v2.10.

Mean cells counts

Concentration [mg/L] (nominal)	Cell counts (* 10^4 cells/mL) at
	24 h	48 h	72 h	96 h
0	2.9	12	45	140
63	2.0	8.2	40	140
125	1.1	8.3	30	110
250	1.3	6.8	28	93
500	0.78	4.6	27	88
1000	0.93	3.5	18	61

Measured test item concentrations

Sample ID	Sodium formate (ppm or %) in samples taken at								Mean
	0 h		96 h						measured
analysis	1	2	A1	A2	B1	B2	C1	C2	concentrations
Control	< 5		< 5						< 5
63 mg/L	62	62	56	56	57	59	57	57	58
125 mg/L	124	125	118	118	121	121	121	121	121
250 mg/L	248	243	227	225	249	249	252	251	243
500 mg/L	505	508	496	494	502	503	469	503	498
1000 mg/L	937	1014	1005	1025	967	1010	1022	1025	1001

Mean measured concentrations were > 80% and < 120% compared to nominal  concentrations.

Remarks on preliminary tests

Two preliminary toxicity tests were conducted to set concentration levels for the definitive test. 

In the first 96 h preliminary test, test concentrations of 1, 10 or 100 mg/L produced growth inhibitions of 0, 18, and 50%, respectively. 

The second preliminary test was initially started as a definitive test with triplicate cultures  at concentrations of 20, 40, 80, 160, and 320 mg/L. Algal cells counts in this study were 110, 110, 110, 110 and 86% of the control population, respectively. The concentration  response was insufficient to define the EC50 and a final definitive test  was set up.

Validity criteria according to OECD 201

Validity of the test

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16.

With the current test it was found to be 53.0. The test fulfills this validity criterion.

 

Evaluation of the section-by-section growth rates:

Arithmetic means of the control replicates from 0 h to 72 h were: Replicate 1: 1.230; Replicate 2: 1.292; Replicate 3: 1.462. [1/d].

Coefficients of variation in control replicates from 0 to 72 h were:

Replicate 1: 5.3%; Replicate 2: 38.7%; Replicate 3: 11.2%.

The mean of the replicate coefficients of variation in the section-by-section growth rate was: 18.4%.

According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The test fulfills this validity criterion.

 

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 9.0%.

According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test does not fulfill this validity criterion! The violation of the latter validiry criterion can be explained by the used methodology for cell count which is relatively inaccurate(microscopy compared to fluorescence). Another reason might be the relatively high titer used at test start.

 

Validity criteria fulfilled:

no

Remarks:

The coefficient of variation of the mean specific growth rate (9%) slightly exceeded the validity criterion (7%), however it is considered that this exceedance does not affect the validity or outcome of the study.

Conclusions:

The 72 h EC20 and EC50 for growth rate were 807 and >1000 mg/L based on nominal exposure concentrations.

Executive summary:

The inhibition of growth of sodium formate to the green algae Pseudokirchneriella subcapitata was determined. The study was carried out according to the following guidelines:

1)       Miller et al. (1978). The Selenastrum capricornutum Printz Algal Assay Bottle Test. Experimental Design, Application and Data Interpretation Research Laboratory, Corvallis, Oregon 97330. EPA-600/9-78-018

2)       OTS Algal Acute Toxicity Test, August, 1982. EG-8, ES-5 and

3)       ASTM (1983). Proposed Standard Practice for Conducting Short-Term Toxicity Tests with Freshwater and Saltwater Algae. E-47.01, Draft No. 5.

The test material was tested using nominal exposure concentrations of; 0 (control), 63, 125, 250, 500 and 1000 mg/L. The overall (0 and 72 h) mean measured concentrations were <5 (control), 58, 121, 243, 498 and 1000 mg/L. The results of the study were therefore based on the nominal concentrations. The 72 h EC20 and EC50 for growth rate were 807 and >1000 mg/L based on nominal exposure concentrations. 

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Study period:

Test performed from 08 Nov 1994 to 11 Nov 1994.

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Guideline study under GLP conditions, but test item concentrations not verified analytically and purity of test substance not known

Justification for type of information:

It is considered appropriate to address the toxicity to aquatic algae and cyanobacteria data requirement for calcium formate by read-across to an available study on potassium formate.

Calcium formate and potassium formate are both salts of formic acid and are members of the Formates Category. For aquatic toxicity endpoints, the category is appropriate for formic acid and its salts because salts immediately dissociate in water to formic acid and counter ions. It is therefore expected that the ecotoxicological profiles of potassium formate and calcium formate will be similar.

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

GLP compliance:

yes

Specific details on test material used for the study:

Potassium formate is being used as a read-across for calcium formate.

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Strain: 1077/5
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa
- Age of inoculum (at test initiation): from exponentially  growing pre-culture, set up three to four days prior to test start
- Method of cultivation: Algae culture maintained at 20±1 °C under continuous white light in the range 6000 to 10000 lux. The culture was continuously shaken.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 ± 1 °C

pH:

test start: 8.0
test end: 9.0

Nominal and measured concentrations:

nominal: 0 (control), 56, 100, 320, 560, and 1000 mg/L (test material)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250-mL concial flasks; 200 mL test volume
- Aeration: none, but incubation on orbital shaker for 72 h
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 1.0E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes (nutrient  stock solutions according to ISO standards and recommendations of VKI  were then added and the pH adjusted to 8.0±0.2)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater, filtered, sterilized (120 °C, 30 min), aerated (2 h)


OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: white light with 6000 - 10000 lx
- Salinity (for marine algae):


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell density after 0, 24, 48, and 72 h (measured using a  microscope and a haemocytometer)
- Other: pH at test start and test end
- endpoints: inhibition of biomass (EbC50; area under the curve) after 72  h and growth rate (ErC50) between 0 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(biomass integral)

Details on results:

NOEC not stated

Cell density

Concentration (mg/L)	Cell density (cells/mL * 10^4)
	0 h	24 h	48 h	72 h
0	1.0	2.8	33.6	95.1
56	1.0	3.4	30.9	92.3
100	1.0	3.7	42.1	70.2
320	1.0	2.7	36.4	92.7
560	1.0	3.7	39.6	86.4
1000	1.0	4.2	27.0	73.6

 

 

Growth inhibition

Concentration	Biomass		Growth rate
(mg/L)	% inhibition		% inhibition
	48h	72h	48h	72h
56	4	4	2	1
100	-28	4	-6	7
320	-7	-2	-2	1
560	-21	-3	-5	2
1000	10	20	6	6

 

Conclusions:

Although the purity of the test material is not known, it is considered that the study is able to sufficiently demonstrate that marine algae are not more sensitive to formate than freshwater algae. This result is considered appropriate for read-across to calcium formate.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Jan 10, 1990 to Jan 14, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

It is considered appropriate to address the toxicity to aquatic algae and cyanobacteria data requirement for calcium formate by read-across to an available study on sodium formate.

Calcium formate and sodium formate are both salts of formic acid and are members of the Formates Category. For aquatic toxicity endpoints, the category is appropriate for formic acid and its salts because salts immediately dissociate in water to formic acid and counter ions. It is therefore expected that the ecotoxicological profiles of sodium formate and calcium formate will be similar.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Procedure for the static algal assay as described in ABC Protocol  #8004-PMN, following methods formulated by:
 
1) Miller et al. (1978). The Selenastrum capricornutum Printz Algal Assay Bottle Test. Experimental Design, Application and Data Interpretation Research Laboratory, Corvallis, Oregon 97330. EPA-600/9-78-018.
2) OTS Algal Acute Toxicity Test, August, 1982. EG-8, ES-5.
3) ASTM (1983). Proposed Standard Practice for Conducting Short-Term Toxicity Tests with Freshwater and Saltwater Algae. E-47.01, Draft No. 5.

GLP compliance:

yes

Remarks:

according to Quality Assurance Officer

Specific details on test material used for the study:

Sodium formate is being used as a read-across for calcium formate.

Analytical monitoring:

yes

Details on sampling:

- Sampling method: 100 mL was collected at test start and after 96 h and in duplicate from control and each concentration.
- Sample storage conditions before analysis: samples were frozen and shipped to Hoechst Celanese Corp.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ABC Laboratories Inc. culture #W
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas, Austin
- Age of inoculum (at test initiation): Algae cultures for the toxicity test were 5 days old at test initiation.
- Method of cultivation: Algae were culivated in tubes containing ca. 10 mL sterile synthetic algae medium. Flasks were 
incubated and agitated (100 rpm) 


ACCLIMATION
- Culturing media and conditions (same as test or not): 100 mL of sterile synthetic algae medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 °C

pH:

6.9 - 7.4 at test start to 7.8 - 8.1 at test end (96 h); (no concentration dependence)

Nominal and measured concentrations:

nominal: 0 (control), 63,  125, 250, 500, and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000-mL test flasks  
- Type (delete if not applicable): Flasks were closed with a foam plug
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: actual mean contol cell density of 8.5 x  10^3 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes; Sterile algae nutrient medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: To 7.5 +- 1 using 1N NaOH
- Photoperiod: continuous
- Light intensity and quality:  4167-4500 lx ("cool white")

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Cell counts were conducted daily for each test replicate using a hemacytometer and compound microscope.
- Concentration of test material in the media was determined at the beginning and end of the incubation period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 20, 40, 80, 160, and 320 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Algal cells counts in this study were 110, 110, 110, 110 and 86% of the control  population, respectively

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistical re-evaluation

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

807 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistical re-evaluation

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistical re-evaluation

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

63 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: original value

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

570 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: original value

Details on results:

- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

Original evaluation:
Cell counts for each replicate and controls  were subjected to analysis of variance (ANOVA) followed by Dunnett's test accepting p < 0.05 
as significant.
EC50 values were calculated from a regression plot. 
Two regression plots were constructed using either the mean cell count or the log of the cell count. 
The regression equation giving the best fit was used to determine the EC50.
Re-evaluation: The raw data for cell count was used to re-evaluate using ToxRat v2.10.

Mean cells counts

Concentration [mg/L] (nominal)	Cell counts (* 10^4 cells/mL) at
	24 h	48 h	72 h	96 h
0	2.9	12	45	140
63	2.0	8.2	40	140
125	1.1	8.3	30	110
250	1.3	6.8	28	93
500	0.78	4.6	27	88
1000	0.93	3.5	18	61

Measured test item concentrations

Sample ID	Sodium formate (ppm or %) in samples taken at								Mean
	0 h		96 h						measured
analysis	1	2	A1	A2	B1	B2	C1	C2	concentrations
Control	< 5		< 5						< 5
63 mg/L	62	62	56	56	57	59	57	57	58
125 mg/L	124	125	118	118	121	121	121	121	121
250 mg/L	248	243	227	225	249	249	252	251	243
500 mg/L	505	508	496	494	502	503	469	503	498
1000 mg/L	937	1014	1005	1025	967	1010	1022	1025	1001

Mean measured concentrations were > 80% and < 120% compared to nominal  concentrations.

Remarks on preliminary tests

Two preliminary toxicity tests were conducted to set concentration levels for the definitive test. 

In the first 96 h preliminary test, test concentrations of 1, 10 or 100 mg/L produced growth inhibitions of 0, 18, and 50%, respectively. 

The second preliminary test was initially started as a definitive test with triplicate cultures  at concentrations of 20, 40, 80, 160, and 320 mg/L. Algal cells counts in this study were 110, 110, 110, 110 and 86% of the control population, respectively. The concentration  response was insufficient to define the EC50 and a final definitive test  was set up.

Validity criteria according to OECD 201

Validity of the test

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16.

With the current test it was found to be 53.0. The test fulfills this validity criterion.

 

Evaluation of the section-by-section growth rates:

Arithmetic means of the control replicates from 0 h to 72 h were: Replicate 1: 1.230; Replicate 2: 1.292; Replicate 3: 1.462. [1/d].

Coefficients of variation in control replicates from 0 to 72 h were:

Replicate 1: 5.3%; Replicate 2: 38.7%; Replicate 3: 11.2%.

The mean of the replicate coefficients of variation in the section-by-section growth rate was: 18.4%.

According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The test fulfills this validity criterion.

 

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 9.0%.

According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test does not fulfill this validity criterion! The violation of the latter validiry criterion can be explained by the used methodology for cell count which is relatively inaccurate(microscopy compared to fluorescence). Another reason might be the relatively high titer used at test start.

 

Validity criteria fulfilled:

no

Remarks:

The coefficient of variation of the mean specific growth rate (9%) slightly exceeded the validity criterion (7%), however it is considered that this exceedance does not affect the validity or outcome of the study.

Conclusions:

The 72 h EC20 and EC50 for growth rate for the green algae Pseudokirchneriella subcapitata when exposed to sodium formate were 807 and >1000 mg/L respectively, based on nominal exposure concentrations. These values are considered appropriate for read-across to calcium formate.

Executive summary:

It is considered appropriate to address the toxicity to aquatic algae and cyanobacteria data requirement for calcium formate by read-across to an available study on sodium formate. Calcium formate and sodium formate are both salts of formic acid and are members of the Formates Category. For aquatic toxicity endpoints, the category is appropriate for formic acid and its salts because salts immediately dissociate in water to formic acid and counter ions. It is therefore expected that the ecotoxicological profiles of sodium formate and calcium formate will be similar.

The inhibition of growth rate of the green algae Pseudokirchneriella subcapitata was determined according to the following guidelines:

1)       Miller et al. (1978). The Selenastrum capricornutum Printz Algal Assay Bottle Test. Experimental Design, Application and Data Interpretation Research Laboratory, Corvallis, Oregon 97330. EPA-600/9-78-018

2)       OTS Algal Acute Toxicity Test, August, 1982. EG-8, ES-5 and

3)       ASTM (1983). Proposed Standard Practice for Conducting Short-Term Toxicity Tests with Freshwater and Saltwater Algae. E-47.01, Draft No. 5.

The test material was tested using nominal exposure concentrations of; 0 (control), 63, 125, 250, 500 and 1000 mg/L. The overall (0 and 72 h) mean measured concentrations were <5 (control), 58, 121, 243, 498 and 1000 mg/L. The 72 h EC20 and EC50 for growth rate were 807 and >1000 mg/L respectively, based on nominal sodium formate exposure concentrations. These values are therefore also considered for calcium formate.

Description of key information

With high probability acutely not harmful to aquatic algae.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

500 mg/L

Additional information

No data are available for calcium diformate therefore a read across was performed to sodium formate.

In a 96-h GLP static algal toxicity study, the 72-h NOEC (growth rate) was determined to be 500 mg/L (Hoechst Celanese, 1990; statistical re-evaluation: ECT, 2010). The 72-h ErC20 was 807 mg/L and the 72-h ErC50 was > 1000 mg/L. Test item concentrations were analytically verified.

It can be concluded with high probability that sodium formate and hence calcium diformate are acutely not harmful to aquatic algae.