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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:
30th July 2012 - 25th October 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 96 hours for quantitative analysis.
- Sample storage conditions before analysis: Samples stored at approximately -20 °C prior to analysis
Vehicle:
no
Details on test solutions:
For the purpose of the definitive test, the test item was dissolved directly in culture medium. Prior to use the test item was heated to 50 °C in order to aid weighing. All test concentrations were corrected for a test item water content
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): N/a


ACCLIMATION
- Acclimation period: - Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
A re-growth test was performed after 96 hours to determine the algicidal or algistatic effect of the test item. Aliquots were removed from each replicate control and test culture (0.25 mL and 0.50 mL respectively) and the replicates pooled. Fresh sterile culture medium (100 mL) was added to each to ensure that the test concentration was reduced to below the inhibiting level.

The flasks were plugged with polyurethane foam bungs are incubated (INFORS Multitron® Version 2 Incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 144 hours.
Test temperature:
24 ± 1 ºC
pH:
Control: from pH 7.8 at 0 hours to pH 8.3 at 96 hours.
Test concentrations: concentration dependent pH 7.6 at 10 mg ai/L through to pH 5.2 at 160 mg ai/L at 0h. See Table 2 in 'Other information on results'
Nominal and measured concentrations:
Control: 10 mg ai/L: 74-91%
20 mg ai/L: 84-93%
40 mg ai/L: 99%
80 mg ai/L: 97-107%
160 mg ai/L: 100-105%
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Material, size, headspace, fill volume: glass, 250mL, fill volume = 100mL
- Aeration: Shaken
- Initial cells density: 1 x 104 cells per mL
- Control end cells density: 2.31 x 106 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/a

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: N/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Intervals of water quality measurement: at 0h and 96h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, culture media had pH adjusted but not test media
- Photoperiod: continuous lighting
- Light intensity and quality: approximately 7000 lux (380-730 nm)
- Salinity (for marine algae): N/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Coulter® Multisizer Particle Counter.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline: N/a
- Range finding study
- Test concentrations: 1, 10, 100, 1000 mg ai/L
- Results used to determine the conditions for the definitive study: No effect on growth at 1 and 10 mg ai/L but growth reduced at 100 and 1000 mg ai/L
Reference substance (positive control):
yes
Remarks:
zinc chloride
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence intervals: 40-52 mg ai/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): cell debris was observed to be present in the test cultures at 160 mg ai/L
- 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: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 96-Hour test period all control, 10, 20 and 40 mg ai/L test cultures were observed to be green dispersions whilst the 80 and 160 mg ai/L test cultures were observed to be clear colorless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 96h EC50 based on growth rate = 0.35 mg/L (95% confidence intervals = 0.3 - 0.4 mg/L)
- Other:
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table 1. 72h and 96h endpoints based on growth rate, yield and biomass inhibition

Time Point

(Hours)

Response Variable

EC50(mg ai/L)

95% Confidence Limits (mg ai/L)

No Observed Effect Concentration (NOEC) (mg ai/L)

Lowest Observed Effect Concentration (LOEC) (mg ai/L)

72

Growth Rate

46

40

-

52

10

20

Yield

20

19

-

21

10

20

Biomass

21

18

-

24

10

20

96

Growth Rate

59

54

-

65

20

40

Yield

38

34

-

42

20

40

Biomass

30

27

-

33

10

20

Table 2. Cell densities and pH measurements in control and test concentrations at 0h and 96h.

Nominal Concentration

(mg ai/L)

pH

Cell Densities*(cells per mL)

pH

0 h

0 h

24 h

48 h

72 h

96 h

96 h

Control

R1

7.8

1.02E+04

4.11E+04

2.17E+05

1.32E+06

2.16E+06

8.3

 

R2

1.06E+04

5.48E+04

3.70E+05

1.53E+06

2.45E+06

 

R3

1.03E+04

5.19E+04

3.50E+05

1.63E+06

2.28E+06

 

R4

9.55E+03

6.12E+04

3.45E+05

1.57E+06

2.33E+06

 

R5

1.03E+04

4.24E+04

3.25E+05

1.46E+06

2.32E+06

 

R6

1.03E+04

6.08E+04

3.88E+05

1.75E+06

2.34E+06

 

Mean

1.02E+04

5.20E+04

3.32E+05

1.54E+06

2.31E+06

10

R1

7.6

1.00E+04

4.67E+04

2.30E+05

1.79E+06

3.33E+06

8.3

 

R2

1.02E+04

4.52E+04

2.19E+05

1.82E+06

3.44E+06

 

R3

1.03E+04

4.70E+04

2.10E+05

1.80E+06

4.12E+06

 

Mean

1.02E+04

4.63E+04

2.19E+05

1.80E+06

3.63E+06

20

R1

7.2

1.05E+04

3.60E+04

1.54E+05

7.67E+05

2.86E+06

8.4

 

R2

1.03E+04

3.63E+04

1.64E+05

8.09E+05

2.55E+06

 

R3

1.00E+04

3.45E+04

1.63E+05

8.00E+05

2.77E+06

 

Mean

1.03E+04

3.56E+04

1.60E+05

7.92E+05

2.73E+06

40

R1

6.5

1.01E+04

2.13E+04

7.11E+04

2.69E+05

9.98E+05

8.0

 

R2

1.00E+04

2.15E+04

6.14E+04

2.33E+05

1.04E+06

 

R3

9.84E+03

2.20E+04

7.39E+04

2.65E+05

1.16E+06

 

Mean

9.99E+03

2.16E+04

6.88E+04

2.56E+05

1.07E+06

80

R1

5.8

1.02E+04

1.83E+04

1.76E+04

1.59E+04

4.83E+04

5.2

 

R2

1.02E+04

1.63E+04

1.88E+04

1.64E+04

2.35E+04

 

R3

1.07E+04

1.89E+04

1.56E+04

1.52E+04

2.53E+04

 

Mean

1.03E+04

1.78E+04

1.74E+04

1.58E+04

3.23E+04

160

R1

5.2

9.78E+03

1.02E+04

6.83E+03

6.31E+03

7.71E+03

4.6

 

R2

9.89E+03

8.18E+03

6.32E+03

8.45E+03

4.87E+03

 

R3

1.01E+04

9.10E+03

7.68E+03

8.83E+03

8.45E+03

 

Mean

9.91E+03

9.17E+03

6.94E+03

7.86E+03

7.01E+03


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

R1- R6= Replicates 1 to 6

Validity criteria fulfilled:
yes
Executive summary:

A 72h ErC50 value of 46 mg active ingredient/L and a NOEC of 10 mg active ingredient/L have been reported for the effects of the test substance on the growth rate of the freshwater alga, Pseudokirchneriella subcapitata based on nomical concentrations. Despite the low pH values in the highest test concentrations (80 and 160 mg ai/L), this study met the OECD 201 validity criteria and has therefore been assigned a reliability rating of 2.

Description of key information

Reliable test results of 72 hour EC50 and NOEC of 46 and 10 mg active acid/l, respectively have been determined for the effect of the HEBMP sodium salt on the growth rate of Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:
46 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

72 hour EC50 and NOEC values of 46 and 10 mg active acid/L have been reported for the effects of HEBMP sodium salt on the growth rate of freshwater alga, Pseudokirchneriella subcapitata, based on nominal concentrations. Whilst the proportion of cyclic and linear constituents was not measured for the tested sample, it is conservatively interpreted that the cyclic constituent could have been present at ca. 50% w/w.

It is a functional property of phosphonate substances that they form stable complexes (ligands) with metal ions. In algal toxicity tests essential nutrients will thus be bound to the phosphonates according to the Ligand binding model [1] (Girling et al. 2018). In algal growth medium some metals form strongly-bound complexes and others form weakly-bound ones (Girling et al. 2018). The phosphonates possess multiple metal-binding capacities, and pH will affect the number of binding sites by altering the ionisation state of the substance. However, the phosphonate ionisation is extensive regardless of the presence of metals (Girling et al. 2018).

The phosphonate-metal complexes may be very stable due to the formation of ring structures ("chelation"). This behaviour ensures that the phosphonic acids effectively bind and hold the metals in solution and renders them biologically less available As a result when a trace metal is complexed, its bioavailability is likely to be negligible (Girling et al. 2018, SIAR 2005). However, there is no evidence of severe toxicity from metal complexes of the ligands (Girling et al. 2018).

In algal growth inhibition tests, complexation of essential trace nutrients (including Fe, Cu, Co, and Zn) by phosphonate substances can lead to inhibition of cell reproduction and growth. Guidelines for toxicity tests with algae do not typically describe procedures for mitigating against this behaviour. For example, the standard OECD Guideline 201, describing the algal growth inhibition test, only specifies that the “chelator content” should be below 1 mmol/l in order to maintain acceptable micronutrient concentrations in the test medium (SIAR 2005).

OECD guidance on the testing of difficult substances and mixtures (OECD, 2000) does include an annex describing “toxicity mitigation testing with algae for chemicals which form complexes with and/or chelate polyvalent metals”. The procedure is designed to determine whether it is the toxicity of the substance or the secondary effects of complexation that is responsible for any observed inhibition of growth. It involves testing the substance in its standard form and as its calcium salt in both standard algal growth medium and in medium with elevated CaCO3 hardness. Calcium is non-toxic to aquatic organisms and does not therefore influence the result of the test other than by competitively inhibiting the complexation of nutrients (SIAR 2005). By increasing the calcium content it may be that the nutrient metals are released from their complexed form although this may not always apply. The outcome of the test however only determines whether nutrient complexation is the cause of apparent toxicity and does not determine the inherent toxicity of the test substance for the reasons explained by the Ligand binding model (Girling et al. 2018).

The magnitude of the stability constants depends on the properties of the metal and also of the ligand, in respect of the type of bonding, the three dimensional shape of the complexing molecule, and the number of complexing groups. The SIAR provides two tables of stability constants (effectively the strength of the complexation), one from Lacour et al. (1999) and one from Gledhill and Feijtel (1992). The Gledhill and Feijtel constants show a range of values for important divalent metal ions, cited as having been obtained from Monsanto internal reports (Owens, 1980). They show that aminomethylenephosphonates are strong complexing agents, with stability constant values ranging from 5 to 24 (Log10 values) and the structural features which constituents of HEBMP share with them makes this conclusion applicable also to HEBMP and its salts.

The complexation constant for phosphonates with iron (III) has been estimated by TNO (1996a) to be around log K = 25 (Girling et al. 2018).

Calculations based on the known stability constants show that, even where the OECD-recommended approach to add additional calcium to the test media is used, that the key nutrients would still be complexed by the phosphonates in preference to complexation of calcium and magnesium. Therefore the calcium complex (most representative of the environmental species) can never be maintained in the test medium in the presence of other key nutrient ions such as Co, Zn, Mn and Fe (Girling et al. 2018). As a result the complexed nutrients will almost certainly not be bioavailable to aquatic plants and this can lead to inhibition of algal growth. Growth inhibition via this mechanism is a secondary effect and does not reflect the inherent toxicity of the test substance (Girling et al. 2018).

The available evidence suggests that toxic effects observed in the tests are a consequence of complexation of essential nutrients and not of true toxicity (SIAR 2005).A study designed to ensure adequate levels of bioavailable nutrients with either of the phosphonates would result in the test substance being a phosphonates-Fe complex. Under conditions where iron is readily available to counteract the effects of nutrient complexation it is unlikely that the substance would have a negative effect on algal growth (Girling et al. 2018). The nutrient complexing behaviour of phosphonate substances therefore renders testing to determine their intrinsic toxicity to algae impractical.

A detailed interpretation of the effects of nutrient complexation, and photolytic release of phosphorus from, phosphonic acids on algal growth in toxicity studies is given in Annex V to the phosphonic acid SIARs (2005). The principal and somewhat contrasting conclusions of the review are that:

   

1) Algal growth may be stimulated by the presence of supplementary phosphorous released by the photolytic degradation of phosphonic acids . 2) Algal growth may be inhibited by the complexation of micronutrients (trace metals) by phosphonic acids. This inhibition is an algistatic rather than algicidal effect. Under the standard test conditions used for most studies, the trace metals will be fully and strongly bound to the HEBMP, with the strong possibility that their bioavailability will have been reduced considerably.

These two phenomena can occur at different stages in the course of the same algal test and at different exposure levels of the substance.

Complexing agents, such as HEBMP, inhibit algal growth as a consequence of their capacity to limit the bioavailability of trace metal micronutrients that are essential for growth. This has been illustrated by the following studies.

Hanstveit and Oldersma (1996) have conclusively demonstrated the importance of taking chelation/complexation into account in tests with another phosphonic acid, DTPMP. They have shown that DTPMP exhibits apparent toxicity (95-h ErC50 = 0.45 mg/l) to Selenastrum capricornutum in growth inhibition tests. These tests were carried out using standard OECD growth medium containing concentrations of Cu, Co and Zn that had been increased above the guideline concentration (Cu: up to 30 times, Co: up to 30x and Zn: up to 300x) in line with their predicted speciation. However, when the test medium was also supplemented with Fe at up to 300x the guideline concentration, no growth inhibition was observed at the highest test concentration of 10 mg/l. The explanation given for the absence of toxicity was that the addition of the Fe ensured that the free ion concentrations of all four of these essential nutrients were now in accordance with those specified in the standard OECD medium as being necessary for healthy algal growth. The experimental concept was for the iron to preferentially bind the DTPMP. The key role of Fe in determining the free concentration of the other elements was based on speciation calculations and an estimated value of the DTPMP-iron stability constant.

Similar findings have been reported by Schowanek et al (1996) from algal toxicity tests carried out with the unrelated chelating substance [S,S]-ethylene diamine disuccinate ([S,S]-EDDS). Chlorella vulgaris was tested according OECD 201, water hardness and trace metal concentrations were varied. In standard media with different water hardness (24-375 mg/l CaCO3), addition of 1 mg/l [S,S]-EDDS reduced the growth rate by 53%, independent on the water hardness. Speciation calculations showed that in the standard medium [S,S]-EDDS is mainly associated with Zn, Cu, and Co. To test the hypothesis that the apparent toxicity was caused by nutrient deficiency, growth experiments in metal-enriched medium were performed. With increasing concentrations of Zn, Co, and Cu, the algal growth increased, reaching a maximum and then falling. The maximum growth was obtained with 1 mg/l (= 3.4 µM) [S,S]-EDDS, 0.62 µM Co, 0.051 µM Cu, and 2.9 µM Zn, where the levels of free Cu, Co and Zn were the same as in standard medium without the chelator. With lower [S,S]-EDDS concentrations, growth is decreased, mainly caused by Zn toxicity.

The interpretation of these data is also consistent with findings presented in the risk assessment being carried out for the chelating agent EDTA (in draft), which is actually a weaker complexing agent than HMDTMP. It has been demonstrated that for EDTA it is not the absolute concentration, but rather the ratio of the EDTA concentration to that of the metal cations that is crucial to determining algal growth under the conditions of a toxicity test (EC, 2003).

The ability of iron to catalyse photodegradation of HMDTMP means that the interpretation of algal growth data can be somewhat uncertain; this applies to the complexing agents discussed above including EDTA. However, limitation of micronutrient availability is considered to be a sufficiently generic phenomenon to explain effects observed in toxicity tests with substances that have the capacity to chelate cationic metals.

Conclusions: Great care must be exercised in interpreting the results of the algal tests carried out with phosphonic acids. The significant potential for nutrient complexation by HEBMP and/or release of phosphorus from degradation of HEBMP to respectively either inhibit or stimulate algal growth makes definitive interpretation difficult. However, the available evidence suggests that toxic effects observed in tests with structurally analogous substances are a consequence of complexation of essential nutrients and not of true toxicity. Therefore, further algal toxicity studies are not recommended  because the results would not provide additional information of the toxicity of HEBMP to algae.

[1] Ligand’ is a general term used to describe a molecule that bonds to a metal; in the present case the phosphonate can form several bonds and the resultant chelated complex can be a very stable entity. It is possible that two molecules could bind to the individual metal, or that one molecule could bind two metals. In dilute solution a 1:1 interaction is the most probable. To simplify discussion, the ligand is able to form a strongly-bound complex with some metals, and a more weakly-bound complex with others.