Iron orthophosphate

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:

The study was conducted between 27 October 2011 and 11 November 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/l) of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Verification of test concentrations
Samples were taken from the control and the 100% v/v saturated solution test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. All 0-Hour samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.
The method of analysis, recovery and test solution analyses are described in the attached Appendix 5.

Vehicle:

no

Details on test solutions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.oC
The culture medium is defined in the attached Appendix 3.


Pre-study media preparation trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Furthermore the test item was shown to be insoluble in the most commonly used solvents.
Based on this information the test item was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions (see attached Appendix 4).

Range-finding test
The results obtained from the pre-study media preparation trial conducted indicated that the use of a saturated solution method of preparation was most appropriate for this test item.
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (7.6 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Samples were taken for chemical analysis from each test concentration at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°C prior to analysis.

Definitive test
Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration no effect on algal growth was observed

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/ml.
A positive control (Harlan Laboratories Ltd Project Number: 41104038) used potassium dichromate as the reference item. Details of the positive control are given in the attached Appendix 2.

The positive control was conducted between 10 October 2011 and 13 October 2011.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

Physico-chemical measurements
The pH of the control and 100% v/v saturated solution test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

The pH value of the control cultures (see Table 2 see in any other information on results) was observed to increase from pH 8.1 at 0 hours to pH 8.5 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration no effect on algal growth was observed.

Details on test conditions:

Experimental Preparation
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (10.2 ml) to give the required test concentration of 100% v/v saturated solution.
The concentration and stability of the test item in the test solutions were verified by chemical analysis at 0 and 72 hours (see Appendix 5).

Exposure conditions
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of solution were used for the control and 100% v/v saturated solution treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.88 x 10 e5 cells per ml. Inoculation of 1 litre of test medium with 10.2 ml of this algal suspension gave an initial nominal cell density of 5 x 10 e3 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron- Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits not stated

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits not stated

Details on results:

RESULTS
Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 72 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0053 mg/l. This does not infer that no test item was in solution, just that which was in solution, was present at a concentration of less than 0.0053 mg/l.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 190 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 5.09 x 103 cells per ml
Mean cell density of control at 72 hours : 9.68 x 105 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data
From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a test concentration of 100% v/v saturated solution over the 72 Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErC10 (0 - 72 h) : >100% v/v saturated solution
ErC20 (0 - 72 h) : >100% v/v saturated solution
ErC50 (0 - 72 h) : >100% v/v saturated solution
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of yield
EyC10 (0 - 72 h) : >100% v/v saturated solution
EyC20 (0 - 72 h) : >100% v/v saturated solution
EyC50 (0 - 72 h) : >100% v/v saturated solution
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences (P0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Physico-chemical measurements
The pH values of the control and 100% v/v saturated solution test preparations are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.

The pH value of the control cultures (see Table 2) was observed to increase from pH 8.1 at 0 hours to pH 8.5 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Verification of test concentrations
Analysis of the test preparations at 0 and 72 hours (see attached Appendix 5) showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0053 mg/l. This does not infer that no test item was in solution, just that which was in solution, was present at a concentration of less than 0.0053 mg/l.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 41104038) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.4 mg/l, 95% confidence limits 1.2 – 1.7 mg/l
EyC50 (0 – 72 h) : 0.59 mg/l, 95% confidence limits 0.53 – 0.65 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.71E+03	1.28E+06
	R2	7.15E+03	1.11E+06	-	-
	Mean	6.93E+03	1.20E+06
0.10	R1	6.24E+03	9.28E+05
	R2	7.31E+03	1.23E+06	3	10
	Mean	6.78E+03	1.08E+06
1.0	R1	5.10E+03	1.40E+06
	R2	5.18E+03	1.24E+06	[7]	[11]
	Mean	5.14E+03	1.32E+06
10	R1	6.50E+03	1.25E+06
	R2	5.73E+03	1.38E+06	[4]	[10]
	Mean	6.11E+03	1.31E+06
100	R1	4.50E+03	1.18E+06
	R2	6.22E+03	1.14E+06	[4]	3
	Mean	5.36E+03	1.16E+06

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.1	5.10E+03	2.16E+04	1.88E+05	9.96E+05	8.5
	R2		5.08E+03	3.06E+04	1.95E+05	9.56E+05
	R3		5.01E+03	3.58E+04	1.73E+05	8.37E+05
	R4		5.10E+03	2.46E+04	1.47E+05	9.33E+05
	R5		5.06E+03	3.02E+04	1.81E+05	1.02E+06
	R6		5.18E+03	3.45E+04	2.21E+05	1.07E+06
	Mean		5.09E+03	2.95E+04	1.84E+05	9.68E+05
100	R1	7.9	5.04E+03	1.57E+04	1.34E+05	9.60E+05	8.3
	R2		5.18E+03	1.72E+04	1.23E+05	9.48E+05
	R3		5.27E+03	1.14E+04	1.26E+05	8.27E+05
	R4		5.08E+03	3.13E+04	2.09E+05	9.90E+05
	R5		5.07E+03	1.04E+04	1.22E+05	8.94E+05
	R6		5.00E+03	1.25E+04	1.08E+05	6.25E+05
	Mean		5.11E+03	1.64E+04	1.37E+05	8.74E+05

 

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.061	0.090	0.069
	R2	0.076	0.077	0.066
	R3	0.082	0.066	0.066
	R4	0.066	0.074	0.077
	R5	0.075	0.075	0.072
	R6	0.080	0.077	0.066
	Mean	0.073	0.077	0.069

 

R₁- R₆= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.074		9.91E+05
	R2	0.073		9.50E+05
	R3	0.071		8.32E+05
	R4	0.073	-	9.28E+05	-
	R5	0.074		1.01E+06
	R6	0.075		1.07E+06
	Mean	0.073		9.63E+05
	SD	0.001		8.02E+04
100	R1	0.073	0	9.55E+05
	R2	0.073	0	9.42E+05
	R3	0.071	3	8.22E+05
	R4	0.073	0	9.85E+05
	R5	0.072	1	8.88E+05
	R6	0.067	8	6.20E+05
	Mean	0.072	2	8.69E+05	10
	SD	0.002		1.35E+05

*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be >= 100% v/v saturated solution.
This study showed that there were no toxic effects at saturation.
This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

Executive summary:

Introduction.

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Furthermore the test item was shown to be insoluble in the most commonly used solvents.

A pre-study media preparation trial indicated that the use of a saturated solution method of preparation was most suitable for this test item.

Following a preliminary range-finding test,Pseudokirchneriella subcapitata was exposed to a solution of the test item at concentrations of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solution was prepared by stirring an excess (50 mg/l) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

Exposure of Pseudokirchneriella subcapitatato the test item gave EC₅₀values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be >= 100% v/v saturated solution.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0053 mg/l. This does not infer that no test item was in solution, just that which was in solution, was present at a concentration of less than 0.0053 mg/l.

This study showed that there were no toxic effects at saturation.

Conclusion

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC₅₀values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

Description of key information

 EC50 (72 h) > 100 mg test item/L nominal (measured iron concentration < LOQ, OECD 201, GLP)

NOEC (72 h) ≥ 100 mg test item /L nominal (measured iron concentration < LOQ, OECD 201, GLP)

Additional information

One study is available assessing the toxicity of Iron (III) orthophosphate (CAS 10045-86-0) towards aquatic algae according to the OECD guideline 201 and GLP. Desmodesmus subspicatus was exposed for 72 h to a single loading rate of 100 mg/L of the test item. An analytical monitoring via ICP-MS was performed. The study resulted in an EC50 (72 h) value of > 100 mg/L and a NOEC of 100 mg/L (nominal loading rate). All measured test item concentrations were below the LOQ of 0.0053 mg/L.