Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007-10-19 to 2007-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD guideline 201

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

link to target

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

2007-10-19 to 2007-11-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read-across

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The present analogue approach contemplates Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine (CAS 98679-19-7) as target substance for read across from the source substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4). The read-across approach is used to evaluate the hazardous potential of the target substance with respect to ecotoxicological endpoints and human health for REACH Annex VII.
Based on similar chemical structures, read-across based on different compounds having the same type of effect(s) as described in scenario 2 of the Read-Across Assessment Framework document can be used as a basis for assessment.
The target substance (Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine) and the source substance (Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates) are both organic UVCB substances.
In consequence, read-across can be justified due to the high structural similarities as well as common properties, which will be outlined in detail below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source: Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (EC 279-632-6, CAS 80939-62-4)
The substance is manufactured from the educts phosphorus pentoxide (CAS 1314-56-3, EC 215-236-1), n-hexanol (CAS 111-27-3, EC 203-852-3) and N-tridecyl-tridecanamine, branched and linear (CAS 101012-97-9, EC309-798-8).

Target: Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine (EC 308-859-6, CAS 98679-19-7)
The substance is manufactured from the educts phosphor pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) and bis(2-ethylhexyl)amine (CAS 106-20-7, EC 203-372-4)

Both substances do not contain any impurities which may impact the feasibility of read-across.

3. ANALOGUE APPROACH JUSTIFICATION
Both substances are organic UVCBs and are manufactured from the educts phosphorus pentoxide (CAS 1314-56-3, EC 215-236-1), alcohols and amines, in similar ratios. In the source chemical, the employed alcohols are 2-butanol (CAS 78-92-2, EC 201-158-5) and iso-butanol (CAS 78-83-1, EC 201-148-0), in the target chemical, n-hexanol (CAS 111-27-3, EC 203-852-3) are used. The second slight difference is the amine part. In the source chemical, N-tridecyl-tridecanamine, branched and linear (CAS 101012-97-9, EC309-798-8) is used, and in the target bis(2-ethylhexyl)amine.
So, the reaction products have a similar distribution pattern with regard to varied alkyl phosphate species (mainly dialkyl phosphates).
The used amines differ in an acceptable manner in their alkyl chain length and so molecular weights, i.e. C13 and 381.73 g/mol (CAS 101012-97-9), and C8 and 241.46 g/mol (CAS 106-20-7), which is hence expected to alter their toxicokinetic behaviour slightly but not essentially. Further, they are both di-substituted amines, which is not considered to change during contact with the bodies fluids, as amines are considered to be rather stable functional groups, e.g. hydrolytically stable, and are not considered to be altered to a relevant extent in the body. Both amines are not covalently bound to the phosphates, so they are expected to dissociate into similar dissociation products, too.
Further, the employed alcohols also only differ slightly in their chain lengths, similarly not altering their toxicokinetic behaviour essentially.
Last but not least, both substances show similar physico-chemical properties. They are both liquid at all relevant handling temperatures, both are characterized by negative glass transition temperatures they do not boil but decompose, have similar densities and low vapour pressures indicating that they are no volatile liquids.
Despite the fact that they have differing logPow values and water solubilities which may alter their uptake and distribution through the body, they are nevertheless suitable for read-across for the only relevant endpoint here, i.e. toxicity to algae and cyanobacteria, which will be outlined on basis of their single educts.
As there is not sufficient data on both source and target substance available, and the amine and the alcohol are considered to be the main difference, data on human health relevant endpoints is retrieved from publically available data sources, i.e. ACToR (https://actor.epa.gov/actor/searchidentifier.xhtml), RTECS (http://ccinfoweb.ccohs.ca/rtecs/search.html), and GESTIS (http://gestis.itrust.de/nxt/gateway.dll?f=templates&fn=default.htm&vid=gestisdeu:sdbdeu). Further, ecotoxicological properties were estimated via US EPA EpiSuite ECOSAR Class Program v1.11; for the source amine the representative structure N(CCCCCCCCCCCCC)CC(C)CCCCCCCCCC was chosen. Last but not least, the amines are not covalently bound to the phosphates.
Based on the available date with regard to human health, all three alcohols are not very toxic, not genotoxic, and do most probably possess skin and eye irritating properties. With regard to ecotoxicological properties, both test data and estimated values indicate that n-hexanol is more toxic than both butanols, so read-across may not underestimated the actual hazard and is hence justified.
Data on the amines indicate that both produce severe irritating reactions when applied into the eye or onto the skin. Further, the available toxicity data indicate that both amines are not highly toxic and bear further no genotoxic potential.
With regard to ecotoxicological properties, the estimated values slightly differ. However, as again the source amine is more toxic than the target one, the actual hazard is not underestimated by read-across, and is so also with regard to the amines justified.
So in consequence, taking into account the similar manufacturing process, i.e. identical and similar educts, and so similar reaction products, similar physico-chemical properties of the source and target chemical and similar (eco)toxicological properties, or at least none that may underestimate the actual hazard, of the source and target amines and alcohols, the read-across from Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates to Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine is scientifically justified.

4. DATA MATRIX

Table: Data Matrix, source and target chemical
Endpoint Source: CAS 80939-62-4 Target: CAS 98679-19-7
Physical state at 20°C, 1013 hPa liquid liquid
Glass transition temperature No melting between -100 and 30 °C -72.1 °C (onset)
Glass transitions at -81 and -48°C
Decomposition >250°C before boiling 167.1°C (onset)
Density 0.9186 g/cm³ 0.968 g/cm³
Vapour pressure 5E-5 Pa at 20°C < 6.7 Pa at 20°C
logPow Mono-hexylphosphate = 1.7 -0.61 at 23°C
di-hexylphosphate = 4.3
tridecylamine (linear) = 5.3
di-tridecylamine (linear) = 11.6 (estimated)
Water solubility 37 mg/l (loading of 1 g/L) 17.14 g/l at 20°C
191 mg/l (loading of 10 g/L)
at 20 °C and pH 4.6
Surface tension 37.7 mN/m at 20°C and 1g/l 37.8 mN/m ≤ST ≤ 41.2 mN/m at 20°C and 1g/l
Flash point 135°C at 101.325 kPa 107.5°C at 101.3 kPa


Table: Data Matrix, source and target alcohol
Endpoint Source alcohol: CAS 111-27-3 Target alcohol: CAS 78-92-2 Target alcohol: CAS 78-83-1
Acute oral toxicity LD50 = 710 mg/kg (rat) (RTECS) LD50 = 2193 mg/kg (rat, RTECS) LD50 = 2460 mg/kg (rat, RTECS)
LD50 = 1950 mg/kg (mouse) (RTECS) LD50 = 4893 mg/kg (rabbit, RTECS) LD50 = 3750 mg/kg (rabbit, RTECS)
LD50 = 4420 mg/kg (rat) (RTECS) LD50 = 2054 mg/kg (rat, RTECS) LD50 = 3500 mg/kg (mouse, RTECS)
LD50 = 4000 mg/kg (mouse) (RTECS) LC50 = 6.48 g/kg (5.73 - 7.32, rat, ACToR) LD50 = 71.1 mg/kg (rabbit, RTECS)
LD50 = 3131 mg/kg (rat) (RTECS)
Acute dermal toxicity LD50 = 3100 µl/kg (rabbit, RTECS) LD50 = 3400 mg/kg (rabbit, RTECS) LD50 > 2000 mg/kg (rat, RTECS)
LD50 = 2330 mg/kg (rabbit, RTECS)
Acute toxicity inhalation LC50 > 1060 mg/m³/6h (rat, RTECS) LC50 > 48500 mg/m³/4h (rat, RTECS) LC50 = 8000 ppm/4h (rat, RTECS)
LC50 > 1060 mg/m³/6h (mouse, RTECS) LC50 = 8000 ppm/4h (rat, RTECS) LC50 = 15500 mg/m³/2h (mouse, RTECS)
LC50 > 1060 mg/m³/6h (guinea pig, RTECS) LC50 = 19200 mg/m³/4h (rat, RTECS) LC50 = 2630 mg/m³/4h (rabbit, RTECS)
Skin irritation Mild reaction (rabbit, open irritation test, 95%, 4h, RTECS) No data No data
Mild reaction (rabbit, Draize test, 95%, 4h, RTECS)
Moderate reaction (rabbit, Draize test, 95%, 24h, RTECS)
Severe reaction (rabbit, Draize test, 12ml/6d, RTECS)
Eye irritation No data Severe reaction (rabbit, Draize test) (RTECS) No data
Genetic Toxicity Negative ±S9 (Ames test) (ACToR) Negative ±S9 (Ames test) (ACToR) Negative ±S9 (Ames test) (ACToR)
Negative (Chromosome aberration in vitro & in vivo) (ACToR)
Fish 96-hr LC50 LC50 = 97.7 mg/l (Fathead minnow, ACToR) LC50 = 3670 mg/l (Fathead Minnow, GESTIS) LC50 = 1330 mg/l (Fish, GESTIS)
120.988 mg/L** 778.872 mg/L** 778.872 mg/L**
Daphnid 48-hr LC50 67.585 mg/L** EC50 = 4230 mg/l (Daphnia magna, GESTIS) LC50 = 1030 mg/l (common aquatic species, GESTIS)
394.678 mg/L** EC50 = 1100 mg/l (Daphnids, GESTIS)
394.678 mg/L**
Green Algae 96-hr EC50 47.059 mg/L** 183.651 mg/L** 183.651 mg/L**
Fish ChV 11.601 mg/L** 66.568 mg/L** 66.568 mg/L**
Daphnid ChV 6.298 mg/L** 28.041 mg/L** 28.041 mg/L**
Green Algae ChV 11.884 mg/L** 37.328 mg/L** 37.328 mg/L**
** Neutral Organic SAR (Baseline Toxicity)


Table: Data Matrix, source and target amine
Endpoint Source amine: CAS 101012-97-9 Target amine: CAS 106-20-7
Acute oral toxicity LD50 = 2760 mg/kg (rat, ECHA) LD50 = 800µL/kg (mouse, intraperitoneal) (ACToR, RTECS)
LD50 = 1640mg/kg (rat) (ACToR, RTECS)
Acute dermal toxicity No data LD50 = 1190µL/kg (rabbit) (ACToR, RTECS)
Acute toxicity inhalation No data No data
Skin irritation Skin Corr Cat. 1A (GESTIS) Severe reaction (rabbit, Draize test) (RTECS)
Corrosive (in vivo, rabbit, ECHA) Severe reaction (rabbit, open irritation test) (RTECS)
Mild reaction (rabbit, open irritation test) (RTECS)
Eye irritation C orrosive (in vivo, rabbit, ECHA) Severe reaction (rabbit, Draize test) (RTECS)
Genetic Toxicity Negative ±S9 (Ames test, HPRT assay, ECHA) Negative ±S9 (Ames test) (ACToR)
Fish 96-hr LC50 3.96e-005 mg/L* 0.047 mg/L*
8.48e-007 mg/L** 0.016 mg/L**
Daphnid 48-hr LC50 1.43e-005 mg/L* 0.010 mg/L*
1.16e-006 mg/L** 0.014 mg/L**
Green Algae 96-hr EC50 1.31e-006 mg/L* 0.003 mg/L*
3.3e-005 mg/L** 0.059 mg/L**
Fish ChV 7.84e-008 mg/L* 0.000546 mg/L*
2.34e-007 mg/L** 0.003 mg/L**
Daphnid ChV 3.45e-006 mg/L* 0.00131 mg/L*
1.31e-006 mg/L** 0.004 mg/L**
Green Algae ChV 9.71e-007 mg/L* 0.00134 mg/L*
6.15e-005 mg/L** 0.039 mg/L**
* Aliphatic Amines
** Neutral Organic SAR (Baseline Toxicity)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item was determined in the duplicate test media samples from the loading rates with 1.0-10 mg/L from both sampling times (0 and 72 hours). The samples from the test concentrations below 1.0 mg/L were not analyzed, since these concentrations were below the determined 72-hour NOEC. From the control samples, only one of the duplicate samples was analyzed from the corresponding sampling times.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
For preparation of the water accommodated fraction (WAFs), three individual mixtures of the test item with the loading rates of 1.0, 3.2, and 10 mg/L were prepared by dispersing the nominal amounts of 3.0, 9.6 and 30.0 mg, respectively, in each 3000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 96 hours. The dispersions were stirred for this period to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (0.45 μm; with reduced negative pressure) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAFs with the lowest loading rates of 0.032 to 0.32 mg/L were prepared as dilutions of the WAF with the loading rate of 1 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Gottingen, Gottingen / Germany)
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: in synthetic test water under same conditions as the test, prepared according to the guideline

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

22-23 °C

pH:

8.0 - 9.2

Nominal and measured concentrations:

Nominal: 0.032, 0.1, 0.32, 1.0, 3.2, 10 mg/L
Measured: three lowest concentration were not analyzed; 0.5, 2.9, 8.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Erlenmeyer flasks covered with glass dishes filled with volumes of 25 ml algal suspension, continuously stirred by magnetic stirrers
- Initial cells density: 10.000 algal cells per mL of test medium
- Control end cells density: 935600 algal cells per mL of test medium (biomass was determined by fluorescence measurement and is given as relative fluorescence units. At the start, 10000 algal cells (ml were inoculated corresponding to 2.5 relative fluorescence units. Mean biomass in control after 72 hour was 233.9 relative fluorescence units)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were dissolved in sterile purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes, reported for dilution water
- Photoperiod: continuous illumination
- Light intensity and quality: measured light intensity of about 6600 lux (mean value), range: 6100 to 7130 lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 3 6W/840), installed about 35 cm above the test flasks

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal biomass in the samples was determined by fluorescence measurement (BIOTEK Multi-Detection Microplate Reader, Model FLx8OO)
- Other: In addition, after 72 hours of exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 10 mg/L). The shape and size of the algal cells were examined microscopically in these samples

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Results used to determine the conditions for the definitive study: The enlarged spacing factor of 3.2 between the test concentrations was chosen, since the concentration-effect relationship was flat according to the results of the range-finding test and thus a large concentration range had to be tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing loading rate of 10 mg/L and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at and up to this test concentration were not affected.

Results with reference substance (positive control):

For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2007 (72-h EC50 for the growth rate: 0.92 mg/L, RCC Study No. B38788) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 2000 to 2007: 72-h EC50: 0.71-1.74 mg/L).

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, the calculated average growth rate and the mean yield at the test concentrations were tested for significant differences when compared to the control values by Dunnett's test.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

EC Number:

279-632-6

EC Name:

Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Cas Number:

80939-62-4

Molecular formula:

not applicable

IUPAC Name:

Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item was determined in the duplicate test media samples from the loading rates with 1.0-10 mg/L from both sampling times (0 and 72 hours). The samples from the test concentrations below 1.0 mg/L were not analyzed, since these concentrations were below the determined 72-hour NOEC. From the control samples, only one of the duplicate samples was analyzed from the corresponding sampling times.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
For preparation of the water accommodated fraction (WAFs), three individual mixtures of the test item with the loading rates of 1.0, 3.2, and 10 mg/L were prepared by dispersing the nominal amounts of 3.0, 9.6 and 30.0 mg, respectively, in each 3000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 96 hours. The dispersions were stirred for this period to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (0.45 μm; with reduced negative pressure) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAFs with the lowest loading rates of 0.032 to 0.32 mg/L were prepared as dilutions of the WAF with the loading rate of 1 mg/L.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Gottingen, Gottingen / Germany)
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: in synthetic test water under same conditions as the test, prepared according to the guideline

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

22-23 °C

pH:

8.0 - 9.2

Nominal and measured concentrations:

Nominal: 0.032, 0.1, 0.32, 1.0, 3.2, 10 mg/L
Measured: three lowest concentration were not analyzed; 0.5, 2.9, 8.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Erlenmeyer flasks covered with glass dishes filled with volumes of 25 ml algal suspension, continuously stirred by magnetic stirrers
- Initial cells density: 10.000 algal cells per mL of test medium
- Control end cells density: 935600 algal cells per mL of test medium (biomass was determined by fluorescence measurement and is given as relative fluorescence units. At the start, 10000 algal cells (ml were inoculated corresponding to 2.5 relative fluorescence units. Mean biomass in control after 72 hour was 233.9 relative fluorescence units)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were dissolved in sterile purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes, reported for dilution water
- Photoperiod: continuous illumination
- Light intensity and quality: measured light intensity of about 6600 lux (mean value), range: 6100 to 7130 lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 3 6W/840), installed about 35 cm above the test flasks

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal biomass in the samples was determined by fluorescence measurement (BIOTEK Multi-Detection Microplate Reader, Model FLx8OO)
- Other: In addition, after 72 hours of exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 10 mg/L). The shape and size of the algal cells were examined microscopically in these samples

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Results used to determine the conditions for the definitive study: The enlarged spacing factor of 3.2 between the test concentrations was chosen, since the concentration-effect relationship was flat according to the results of the range-finding test and thus a large concentration range had to be tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing loading rate of 10 mg/L and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at and up to this test concentration were not affected.

Results with reference substance (positive control):

For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2007 (72-h EC50 for the growth rate: 0.92 mg/L, RCC Study No. B38788) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 2000 to 2007: 72-h EC50: 0.71-1.74 mg/L).

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, the calculated average growth rate and the mean yield at the test concentrations were tested for significant differences when compared to the control values by Dunnett's test.