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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2000 - 13 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The purity, expiration date, and lot number of the test substance were not given.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES: DIAMMONIUM HYDROGENORTHOPHOSPHATE
- Melting point: 155°C (decomposition)
- Vapour pressure at 20°C: 7.62 x 10-2 Pa
: 5.71 x 10-4 mmHg
- Water solubility (under test conditions): 400 - 588 g/L

- Henry's law constant: not applicable
- log Pow: not applicable


OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal: 0.0 (control), 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg DAP/L
- Sampling method: At test initiation (0 hrs), 15-mL samples were collected from each parent solution. At test termination (72 hrs), 4-mL samples were removed from each individual replicate and composited by treatment into a 12-mL total sample.
- Sample storage conditions before analysis:no data.
Vehicle:
no
Details on test solutions:
STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Vehicle, solvent: no
- Concentration of vehicle/ solvent: 0 mL/L
- Other procedures: 100 and 50 mg/L were prepared by direct addition to freshwater algal nutrient medium (FWAM) followed by sonication and 50% serial dilutions were prepared from the 50 mg/L solution in FWAM to prepare subsequent test solutions.

DILUTION WATER
- Source: freshwater algal nutrient medium by addition of nutrients to ABC reagent water. After preparation, the algal medium
was pH-adjusted to 7.5 ± 0.1 (using 0.1 N HCl) and filtered through a 0.45-μm Millipore filter.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: Pseudokirchnerella subcapitata
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas at Austin, Austin, Texas
- Age of inoculum (at test initiation): 6 days old culture
- Method of cultivation: In 250-mL Erlenmeyer flasks fitted with foam stoppers. Positioned on a rotary shaker set at 100 rpm and incubated for 72 hours at 24 ± 2°C under continuous cool-white fluorescent lighting. Light intensity was maintained at approximately 8600 ± 860 lux.

ACCLIMATION
- Acclimation period: No data.
- Culturing media and conditions (same as test or not): Freshwater algal nutrient medium (FWAM), same as test. The medium was prepared by the
addition of appropriate reagent grade nutrients to ABC reagent water. After preparation, the medium was pH-adjusted to 7.5 ± 0.1 (using 0.1 N HCl) and filtered through a 0.45-:m Millipore filter.
- Any deformed or abnormal cells observed: No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Cell counts were performed every 24 ± 1 hour after inoculation
Post exposure observation period:
no data
Hardness:
No data
Test temperature:
22.5-25.7 °C
pH:
ranged from 7.39 to 7.42 at test initiation and from 7.56 to 9.35 at test termination
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
- Nominal concentrations (mg/L): 0, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L DAP
- Calculated concentrations at initiation: 0, 1.65, 3.57, 6.41, 12.0, 24.1, 47.4 and 97.1mg/L (95-114% of nominal).
DAP concentrations decreased during the exposure period (43-86% of niminal) which was likely due to incorporation of the phosphate into the algal biomass rather than instability of the test substance. Therefore, effect values were determined based on initial calculated concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flask containing 100 mL solution, fitted with foam stoppers.
- Initial cells density: 1.05 x10*4 cells/mL
- Control end cells density: 103 x10*4 cells/mL
- No. of organisms per vessel: 103 x10*6 cells/vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):3
- No. of vessels per vehicle control (replicates): n/a (no vehicle)

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
Light intensity in the environmental chamber ranged from 8034 to 8788 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were performed every 24 ± 1 hour after inoculation on all replicates of the control and
each test concentration. The algal cell counts were accomplished utilizing a hemacytometer and an optical microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2-fold
- Range finding study
- Test concentrations: nominal test concentrations of 0.10, 1.0, 10, 100, and 1000 mg DAP/L.
- Results used to determine the conditions for the definitive study:
Cell counts for the four lowest test concentrations ranged from 95.0 to 140x10*4 cells/mL. The control cell count was 103x10*4 cells/mL. The cell count in the 1000 mg DAP/L solution was 13.3x10*4 cells/mL. This test solution was cloudy and contained particulate matter throughout the test, indicating that the test substance was not soluble at this concentration.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 97.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
growth rate
Remarks on result:
other: No inhibition of algal growth observed in study
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 97.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
biomass
Remarks on result:
other: No inhibition of algal growth observed
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
97.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: element phosphate
Basis for effect:
growth rate
Remarks on result:
other: Toxicity
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.57 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
growth rate
Remarks on result:
other: Stimulation of algal growth
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.57 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
biomass
Remarks on result:
other: Stimulation of algal growth
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Statistical analysis to determine EbC50 and ErC50 values was not performed due to the lack of inhibition of growth during the definitive test.
The NOEC was calculated using Dunnett’s test as performed by SAS.

none

Validity criteria fulfilled:
yes
Remarks:
Growth factor control: 98
Conclusions:
NOEC (toxicity) = 97.1 mg DAP/L (no inhibition of algal growth observed in study) / NOEC (stimulation) = 3.57 mg DAP/L
No adverse inhibitory effects were observed. There was some growth stimulation at 6.25 mg/L and above. This was consistent with the known stimulatory effect of phosphate compounds. The reported NOEC reflects this stimulatory response and is not considered to be an adverse effect.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Reason / purpose for cross-reference:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 97.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
growth rate
Remarks on result:
other: No inhibition of algal growth observed in study
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 97.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
biomass
Remarks on result:
other: No inhibition of algal growth observed
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
97.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: element phosphate
Basis for effect:
growth rate
Remarks on result:
other: Toxicity
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.57 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
growth rate
Remarks on result:
other: Stimulation of algal growth
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.57 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
phosphate
Basis for effect:
biomass
Remarks on result:
other: Stimulation of algal growth
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Statistical analysis to determine EbC50 and ErC50 values was not performed due to the lack of inhibition of growth during the definitive test.
The NOEC was calculated using Dunnett’s test as performed by SAS.

none

Validity criteria fulfilled:
yes
Remarks:
Growth factor control: 98
Conclusions:
NOEC (toxicity) = 97.1 mg DAP/L (no inhibition of algal growth observed in study) / NOEC (stimulation) = 3.57 mg DAP/L
No adverse inhibitory effects were observed. There was some growth stimulation at 6.25 mg/L and above. This was consistent with the known stimulatory effect of phosphate compounds. The reported NOEC reflects this stimulatory response and is not considered to be an adverse effect.

Description of key information

One key study exists on an analogous substance. This study had been conducted in accordance with a recommended guideline (OECD 201, EU Method C.3) and under the conditions of GLP. As such, no further testing is considered necessary.

Key value for chemical safety assessment

EC50 for freshwater algae:
97.1 mg/L
EC10 or NOEC for freshwater algae:
97.1 mg/L

Additional information

NOEC (toxicity) = 97.1 mg DAP/L (no inhibition of algal growth observed in study) / NOEC (stimulation) = 3.57 mg DAP/L

No adverse inhibitory effects were observed. There was some growth stimulation at 6.25 mg/L and above. This was consistent with the known stimulatory effect of phosphate compounds. The reported NOEC reflects this stimulatory response and is not considered to be an adverse effect.