Reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide

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:

1997-07-01 to 1997-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted June 7, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

December 29, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- For analytical verification of the test substance concentrations at the start of the test duplicate samples were taken from the freshly prepared test media (without algae) of all test concentrations and the control.
- For determination of the stability of the test substance under the test conditions or the maintenance of the test substance concentration during the test period, sufficient volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test (but without algae), and were sampled in duplicate at the end of the test (after the 72-hour test period).
- All samples were deepfrozen (about -20°C) immediately after sampling.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Pretest: A pre-experiment was carried out to determine the solubility of the test substance in test water. The test substance was dissolved in test water up to a concentration of about 500 mg/L (determined analytically by specific measurement of aluminium) at room temperature by stirring for 2-24 hours.
- Method: The test medium of the highest test concentration was therefore prepared as follows: 50 mg of the test item was mixed with test water to a volume of 500 mL, treated in an ultrasonic bath for five minutes, stirred for four hours at room temperature in the dark and again treated in an ultrasonic bath for fifteen minutes. In this way a turbid solution with a concentration of 100 mg/L was obtained. Defined volumes of this intensively stirred test medium were mixed into test water to obtain the test media of the following lower test concentrations: 32, 10, 3.2, and 1.0 mg test substance/L. Immediately prior to test start the algae were inoculated by adding an adequate volume from the pre-culture.
- Controls: Test water without test substance

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: CHODAT, Strain No.86.81 SAG
- Source (laboratory, culture collection): supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", D-37073 Göttingen
- Method of cultivation: The algae were grown in the RCC laboratories under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (=24 mg/L) as CaCO3

Test temperature:

24°C

pH:

The initial pH increased with test concentrations from 8.1 in the controls to 9.7 at 100 mg/L. After 72 hours pH was in the range from 8.5 to 10.7.

Nominal and measured concentrations:

nominal: 0(control), 1.0, 3.2, 10, 32, and 100 mg/L

Details on test conditions:

TEST SYSTEM
- The test was started (hour-0) by inoculation of a biomass of 10.000 algal cells per mL test medium. These cells were taken from a exponentially growing pre-culture, which was set up about three days prior to the test under identical conditions as in the test.
- The test design consisted of three replicates per test concentration and six replicates for the control. Volumes of 50 mL algal suspension (treated and untreated) per replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.
- The flasks were covered with glass dishes, incubated in a temperature-controlled water bath at 24°C, and continuously illuminated at a measured light intensity in the range from 7200 to 8100 Lux (minimum and maximum value of measurements in six places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W-1/84), installed above the water bath in a distance of about 35 cm from the test flasks.
- Because of potential test substance particles in the higher concentrated test media where turbidity was observed two additional replicates per test concentration without algae were incubated together with the test media containing algae as "particle controls". These additional replicates were incubated and handled in the same way as the test media with algae and were used for correction of the algal cell densities.
- Evaluations:
-- pH: The pH-values of the test media were measured in samples from all test concentrations and the control at the start and the end of the test.
-- Temperature: During the test duration the test media temperature was measured daily in Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETER
Analytical grade salts were added to sterile bidistilled water at the following final nominal concentrations:
- Macro-nutrients:
-- NaHCO3: 50.0 mg/L
-- CaCl2 x 2 H2O: 18.0 mg/L
-- NH4Cl: 15.0 mg/L
-- MgSO4 x 7 H2O: 15.0 mg/L
-- MgCl2 x 6 H2O: 12.0 mg/L
-- KH2PO4: 1.6 mg/L
- Trace elements:
-- Na2EDTA x 2 H2O: 100.0 µg/L
-- FeCl3 x 6 H2O: 80.0 µg/L
-- MnCl2 x 4 H2O: 415.0 µg/L
-- H3BO3: 185 µg/L
-- Na2MoO4 x 2 H2O: 7.0 µg/L
-- ZnCl2: 3.0 µg/L
-- CoCl2 x 6 H2O: 1.5 µg/L
-- CuCl2 x H2O: 0.01 µg/L

EFFECT PARAMETERS MEASURED:
- Counting and examination of the algal cells:
-- A small volume of the test media (2.0 mL) was withdrawn from all test flasks and the additional replicates containing the test suspensions without algae ("particle controls") after 24, 48, and 72 hours of exposure and was not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM, three measurements per sample). The additional replicates containing the test suspensions without algae were counted in the exact same way as the test media with algae.
-- In addition, a sample was taken after 72 hours of exposure from control and from the 32 mg/L test concentration (nominal) with reduced algal growth to examine and compare the shape of the treated algal cells microscopically.

- Determination of the algal growth inhibition: Inhibition of algae growth was determined from:
-- The area under the growth curves AUC (=biomass)
-- The specific growth rate µ for exponentially growing cultures

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

µ

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

19.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

µ

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

µ

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: as representative element for the whole substance, aluminium was measured by AAS at initiation and finalisation of exposure

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: as representative element for the whole substance, aluminium was measured by AAS at initiation and finalisation of exposure

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: as representative element for the whole substance, aluminium was measured by AAS at initiation and finalisation of exposure

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

µ

Remarks on result:

other: as representative element for the whole substance, aluminium was measured by AAS at initiation and finalisation of exposure

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

14.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

µ

Remarks on result:

other: as representative element for the whole substance, aluminium was measured by AAS at initiation and finalisation of exposure

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

µ

Remarks on result:

other: as representative element for the whole substance, aluminium was measured by AAS at initiation and finalisation of exposure

Details on results:

MEASURED TEST SUBSTANCE CONCENTRATIONS:
- at start: In the test media the mean measured test substance concentrations at the start of the test ranged from 81 to 92 % of the nominal values.
- at end: During the test period of 72 hours a decrease of test substance concentration in the test media was determined. At the end of the test 50 - 80 % of the nominal values were found. The losses were most probably due to precipitation of the test substance caused by the limited solubility of the test item. This is supported by the fact that the losses were greater at 100 and 32 mg/L nominal than at 10 mg/L nominal. The total mean measured test concentrations (average of all measurements per test concentration) varied in the range from 67 to 87 % of the nominal values.

INFLUENCE OF THE TEST ITEM ON THE GROWTH OF SCENEDESMUS SUBSPICATUS:
- LOEC: After 72 hours of exposure the test substance showed a statistically significant inhibition effect on the growth of Scenedesmus subspicatus starting at the mean measured test substance concentration of 21.6 mg/L (nominal concentration of 32 mg/L). Thus, this test concentration was determined to be the 72-hour LOEC (lowest concentration tested with toxic effects).
- NOEC: The 72-hour NOEC (highest concentration tested without toxic effects) was determined to be at the mean measured test substance concentration of 8.6 mg/L (nominal concentration of 10 mg/L) since up to and including this test concentration both the mean algal biomass and growth rate were not lower by statistically significant extent than in the control.
- EC50/EC10: see above
- Microscopic examination: The microscopic examination of the shape of the algal cells after 72 hours in the second highest nominal test substance concentration of 32 mg/L showed no difference in comparison to the cells in the control. Thus, the shape of the algal cells was not affected by the test item up to and including this concentration.
- Controls: In the control the cell density increased from N= 1x10exp4 cells/mL at the start of the test (hour-0) to N= 119x10exp4 cells/mL (mean value) after 72 hours. Thus, algal growth in the control was sufficiently high under the test conditions.
- pH: At the start of the test, the pH-values in the test media (control to 100 mg/L test concentration) ranged from 8.1 to 9.7 and at the end of the test from 10.7 to 8.5. This increase of the pH in the control and test concentrations without any significant algal growth inhibition over the test period was caused by the CO2-consumption of the algae due to their rapid growth, i.e. high cell densities (despite intense stirring of the test media). The decrease of the pH in the two highest test concentrations of 32 and 100 mg/L was probably an effect of the precipitation of the test item during incubation and is in line with the analytical findings.

Validity criteria fulfilled:

yes

Executive summary:

In a 72-h acute toxicity study according to OECD guideline 201, adopted June 7, 1984 and EU Method C.3, dated December 29, 1992, cultures of Scenedesmus subspicatus (new name: Desmodesmus subspicatus) CHODAT were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" at nominal concentrations of 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L (the three last mentioned concentrations correspond to measured concentrations of 8.6 (86 % of nominal), 21. 6 (67 % of nominal), and 67.2 (67 % of nominal) mg/L, respectively) under static conditions.

The NOEC, EC₅₀, and EC₁₀ values based on measured concentrations and biomass were 8.6 (nominal 10), 16.0 (nominal 21.9), and 9.7 (nominal 12.3) mg/L, respectively.

The NOEC, LOEC, EC₅₀, and EC₁₀ values based on measured concentrations and growth rate µ were 8.6 (nominal 10), 21.6 (nominal 32), 19.9 (nominal 28.9), and 14.6 (nominal 19.7) mg/L, respectively.

The microscopic examination of the shape of the algal cells after 72 hours in the second highest nominal test substance concentration of 32 mg/L showed no difference in comparison to the cells in the control. Thus, the shape of the algal cells was not affected by the test item up to and including this concentration. In the control, the cell density increased from N= 1x10exp4 cells/mL at the start of the test (hour-0) to N= 119x10exp4 cells/mL (mean value) after 72 hours. Thus, algal growth in the control was sufficiently high under the test conditions.

This toxicity study is classified as acceptable and satisfies the guideline requirements for a 72-h algal growth inhibition test.

Description of key information

A reliable, relevant and adequate study according to OECD Guideline 203, adopted April 4, 1984 and EU method C-1, adopted September, 1984 is available.

The NOEC, EC50, and EC10 values for Scenedesmus subspicatus (new name: Desmodesmus subspicatus) based on measured concentrations and growth rate µ were 8.6 (nominal 10), 19.9 (nominal 28.9), and 14.6 (nominal 19.7) mg/L, respectively.
The dissociation of the substance in water will result in common environmental constituents - calcium, aluminium, phosphate. Calcium and phosphate are essential for almost all living organisms including aquatic algae and cyanobacteria and natural constituents of their habitats. Aluminium as most abundant metallic element of the earth´s crust, is also a natural component in environmental habitats. Therefore, the substance is not expected to have a relevant intrinsic toxic activity to aquatic organisms.
Noticed toxicity is most probably triggered by a pH effect. Dissociation of the reaction mass under the conditions of OECD Guidelines 112 or 105, resulted in a pKa of 10.1 and a pH of 11.4, respectively. A temporary pH effect as toxicodynamic reason for aquatic toxicity of inorganic alkaline substances dissolving in common environmental constituents has been discussed in detail in the EU Risk Assessment on sodium hydroxide and in several OECD HPV SIDS documents on alkaline substances. In general, natural waters do have a sufficient buffering capacity to overcome this effect (c. f. EU RAR sodium hydroxide, 2007; http: //echa. europa. eu/documents/10162/0ded9c53-4082-405b-b09a-e16e57e158af) or e. g. http: //www. inchem. org/documents/sids/sids/Naco. pdf). Therefore, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not expected to have a relevant intrinsic toxic activity to aquatic organisms and PNECs will not be derived.

Key value for chemical safety assessment

EC50 for freshwater algae:

19.9 mg/L

EC10 or NOEC for freshwater algae:

8.6 mg/L

Additional information

In a 72-h acute toxicity study according to OECD guideline 201, adopted June 7, 1984 and EU Method C.3, dated December 29, 1992, cultures of Scenedesmus subspicatus (new name: Desmodesmus subspicatus) CHODAT were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) at nominal concentrations of 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L (the three last mentioned concentrations correspond to measured concentrations of 8.6 (86 % of nominal), 21. 6 (67 % of nominal), and 67.2 (67 % of nominal) mg/L, respectively) under static conditions.

The NOEC, EC₅₀, and EC₁₀ values based on measured concentrations and biomass were 8.6 (nominal 10), 16.0 (nominal 21.9), and 9.7 (nominal 12.3) mg/L, respectively.

The NOEC, LOEC, EC₅₀, and EC₁₀ values based on measured concentrations and growth rate µ were 8.6 (nominal 10), 21.6 (nominal 32), 19.9 (nominal 28.9), and 14.6 (nominal 19.7) mg/L, respectively.

The microscopic examination of the shape of the algal cells after 72 hours in the second highest nominal test substance concentration of 32 mg/L showed no difference in comparison to the cells in the control. Thus, the shape of the algal cells was not affected by the test item up to and including this concentration. In the control the cell density increased from N= 1x10exp4 cells/mL at the start of the test (hour-0) to N= 119x10exp4 cells/mL (mean value) after 72 hours. Thus, algal growth in the control was sufficiently high under the test conditions.

This toxicity study is classified as acceptable and satisfies the guideline requirements for a 72-h algal growth inhibition test.