Isopropylamine

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:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (e.g. no analytical verification of test concentrations, non-GLP)

Qualifier:

according to guideline

Guideline:

other: German industrial standard test guideline DIN 38412, part 9

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

re-evaluated in 2007 according to OECD 201 (adopted March 2006) guideline

Deviations:

not applicable

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A stock solution containing 40 mg/L test item was prepared. Test concentrations of 0 (control), 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 and 20 were prepared, each containing 10 000 cells/ml. All test concentrations were prepared in 5 replicates plus 2 blanks for fluorescence measurements (w/o cells) per concentration. The cell density was measued after 0, 24, 48, 72, 96 h. Conductivity and pH were measured in the mixtures.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: SAG 86.81
- Age of inoculum (at test initiation): 72 h

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Test batches with exposure durations of 24, 48, 72 and 96 h were tested.

Test temperature:

20 °C

pH:

see IUCLID section 'Any other information on results incl. tabes'

Dissolved oxygen:

---

Nominal and measured concentrations:

0 (control), 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 mg/L (nominal)

Details on test conditions:

see IUCLID section 'Any other information on materials and methods incl. tables'

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on re-calculation (see below)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on re-calculation (see below)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on re-calculation (see below); 95% CI: 2.41-2.70

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on re-calculation (see below); 95% CI: 4.90-5.28

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on re-calculation (see below); 95% CI: 18.2-19.6

Details on results:

Growth curves (Cell density vs. test item concentration) were given for each of the measured exposure durations (96h, 72h, 48h, 24h) in the original report. During the statistical re-evaluation (ECT Oekotoxikologie GmbH, 2007), a concentration-effect curve showing the influence of the test item on yield of the introduced Desmodesmus subspicatus as observed after 72 h was created (see IUCLID section 'Illustration (picture/graph)' by Probit analysis using linear max. likelihood regression. Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984).
Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively. Slope function after Litchfield and Wilcoxon: 3.325. (The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b)).

Results with reference substance (positive control):

not available

Reported statistics and error estimates:

See IUCLID section "Details on results".

Statistical re-evaluation with ToxRatPro (v2.09): Alga, Growth inhibition test (OECD 201, EEC, ISO); see reference (ECT Oekotoxikologie GmbH, 2007).

The following nominal effect concentrations related to yield (EyC), growth rate (ErC), and biomass integral (EbC) were recalculated according to OECD TG 201 (adopted 23 Mar 2006) using the fluorescence values for inhibition of biomass from the original report. Since no calibration curve data were available, fluorescence data were equated with cell numbers.

Results are based on the arithmetic mean of five replicates for treatments 0.156 to 20 mg/L.

YIELD

Effective Concentrations (ECx) with Yield at 72 and 96 h:

Probit analysis using linear max. likelihood regression was applied for determining the EC10, EC20 and EC50 values for the 72 and 96 h data (95% confidence intervals in brackets).

Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984). Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively. Slope function after Litchfield and Wilcoxon: 3.325 (72 h); 4.179 (96 h).

Threshold Concentrations (NOEC) with Yield at 72 h

Normal distribution of the yield at 72 h was tested with the Kolmogorov-Smirnov-test (goodness of fit; modification after Lilliefors 1967; significance level alpha = 0.05).

Variance Homogeneity was tested with Cochran´s Test Procedure (alpha = 0.05)

Normal distribution and variance homogeneity requirements are fulfilled; a parametric multiple test is advisable.

Williams Multiple Sequential t-test Procedure was applied for determining the NOEC for the 72 h data.

Threshold Concentrations (NOEC) with Yield at 96 h

Normal distribution of the yield at 96 h was tested with the Kolmogorov-Smirnov-test (goodness of fit; modification after Lilliefors 1967; significance level alpha = 0.05).

Variance Homogeneity was tested with Cochran´s Test Procedure (alpha = 0.05)

Variance homogeneity check was not passed but normal distribution requirements are fulfilled; the parametric Bonferroni-t-test for non-homogeneous variances is advisable.

Welch-t test for Inhomogeneous Variances with Bonferroni Adjustment was applied for determining the NOEC for the 96 h data.

Yield (mg/l)
------------------
                                  72 h                          96 h
EyC10:              0.887 (0.725-1.05)   0.221 (0.088-0.370)
EyC20:              1.51 (1.30-1.71)       0.414 (0.214-0.618)
EyC50:              4.13 (3.78-4.51)       1.376 (0.994-1.90)

NOEyC:            0.625                              0.313
LOEyC:             1.25                                 0.625

GROWTH RATE

Effective Concentrations (ECx) with Growth Rate at 72 and 96 h:

Probit analysis using linear max. likelihood regression was applied for determining the EC10, EC20 and EC50 values for the 72 and 96 h data (95% confidence intervals in brackets).

Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984). Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively. Slope function after Litchfield and Wilcoxon: 4.765 (72 h) 9.562 (96 h)

Threshold Concentrations (NOEC) with Growth Rate at 72 h

Normal distribution of the yield at 72 h was tested with the Kolmogorov-Smirnov-test (goodness of fit; modification after Lilliefors 1967; significance level alpha = 0.05).

Variance Homogeneity was tested with Cochran´s Test Procedure (alpha = 0.05)

Variance homogeneity check was not passed but normal distribution requirements are fulfilled; the parametric Bonferroni-t-test for non-homogeneous variances is advisable.

The Welch-t test for Inhomogeneous Variances with Bonferroni Adjustment was applied for determining the NOEC for the 72 h data.

Threshold Concentrations (NOEC) with Growth Rate at 96 h

Normal distribution of the yield at 72h was tested with the Kolmogorov-Smirnov-test (goodness of fit; modification after Lilliefors 1967; significance level alpha = 0.05).

Variance Homogeneity was tested with Cochran´s Test Procedure (alpha = 0.05)

Normal distribution and variance homogeneity requirements are fulfilled; a parametric multiple test is advisable.

Williams Multiple Sequential t-test Procedure was applied for determining the NOEC for the 96 h data.

Growth rate (mg/l)
------------------
                                       72 h                         96 h
ErC10:              2.56 (2.41-2.70)       0.833 (0.604-1.708)
ErC20:              5.09 (4.90-5.28)       2.26 (1.84-2.67)
ErC50:              18.9 (18.2-19.6)       15.0 (12.7-18.2)

NOErC:            1.25                           0.313
LOErC:             2.5                             0.625

BIOMASS INTEGRAL

Effective Concentrations (ECx) with Biomass Integral at 72 and 96 h:

Probit analysis using linear max. likelihood regression was applied for determining the EC10, EC20 and EC50 values for the 72 and 96 h data (95% confidence intervals in brackets).

Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984). Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively. Slope function after Litchfield and Wilcoxon: 3.619 (72 h); 4.434 (96 h).

Threshold Concentrations (NOEC) with Biomass Integral at 72 h

Normal distribution of the yield at 72 h was tested with the Kolmogorov-Smirnov-test (goodness of fit; modification after Lilliefors 1967; significance level alpha = 0.05).

Variance Homogeneity was tested with Cochran´s Test Procedure (alpha = 0.05)

Normal distribution and variance homogeneity requirements are fulfilled; a parametric multiple test is advisable.

Williams Multiple Sequential t-test Procedure was applied for determining the NOEC for the 72 h data.

Threshold Concentrations (NOEC) with Biomass Integral at 96 h

Normal distribution of the yield at 72 h was tested with the Kolmogorov-Smirnov-test (goodness of fit; modification after Lilliefors 1967; significance level alpha = 0.05).

Variance Homogeneity was tested with Cochran´s Test Procedure (alpha = 0.05)

Variance homogeneity check was not passed but normal distribution requirements are fulfilled; the parametric Bonferroni-t-test for non-homogeneous variances is advisable.

The Welch-t test for Inhomogeneous Variances with Bonferroni Adjustment was applied for determining the NOEC for the 92 h data.

Biomass integral (mg/l)
-----------------------
                                      72 h                           96 h
EbC10:               1.13 (0.739-1.52)     0.439 (0.309-0.576)
EbC20:               1.99 (1.47-2.49)       0.846 (0.656-1.04)
EbC50:               5.84 (4.94-6.97)       2.95 (2.56-3.42)

NOEbC:             1.25                          0.313
LOEbC:              2.5                            0.625

Original effect values as given in the report (nominal concentrations in mg/l):

---------------------------------------------------------------------------

	72h	96h
EC20	1.48	0.47
EC50	4.13	1.2
EC90	n.d.	14.2

n.d.: not determined

pH at test start (0 h) and at test end (96 h)
-------------------------------------------

   Conc. [mg]                      0 h                        72 h

                                  uninoculated             uninoculated           inoculated

0 (control)                        8.1                       8.5                         9.7

0.156                                  8.0                        8.4                         9.8

0.313                                  8.0                        8.3                         9.7

0.625                                  8.0                        8.3                        9.6

1.25                                    8.1                        8.2                        9.2

2.50                                    8.2                        8.2                         9.2

5                                         8.3                        8.2                         8.8

10                                       8.6                        8.2                         8.6

20                                       9.0                        8.3                         8.6

Parameters tested in the stock solution:

----------------------------------------

pH: 10.6

conductivity: 73 µS/cm

No data were given on oxygen and temperature development during the test.

VALIDITY CRITERIA

(1) The biomass (cell number) increased exponentially by a factor of at least 16 within the 72 -hour test period. The factor of cell number measured in the control between 0 and 72 h was 64.2, which is > 16.

The testfulfils this validity criterion.

(2) Evaluation of the sectional growth rates (the mean coefficient of variation of sectional growth rates calculated for the individual control replicates must not be higher than 35%):

 Coefficients of variation in control replicates from 0 to 72 h were:

Replicate 1: 12.5%; Replicate 2: 13.9%; Replicate 3: 8.0%; Replicate 4: 12.8%; Replicate 5: 19.2%. The mean of the replicate coefficients of variation in the section-by-section growth rate was: 13.3%.

Regarding the test period of 72 h, the test fulfils this validity criterion.

(3) The coefficient of variation of average specific growth rates in control cultures was 2.3% after 72 h and 1.2 after 96 h, which is < 7%.

The test fulfils this validity criterion.

Validity criteria fulfilled:

yes

Conclusions:

Toxcity of isopropylamine towards freshwater green algae (German industrial standard test guideline DIN 38412, part 9; recalculated according to OECD TG 201 (adopted 23 Mar 2006); GLP):
EC50 (growth rate; 72 h; 95% CI in brackets): 18.9 (18.2-19.6)  mg/L
EC10 (growth rate; 72 h; 95% CI in brackets): 2.56 (2.41-2.70) mg/L
NOEC (growth rate; 72 h): 1.25 mg/L

Executive summary:

The substance isopropylamine was tested for algal toxicity in a reliable study performed compliant with GLP according to the German industrial standard test guideline DIN 38412, part 9. The study was statistically re-evaluated according to OECD 201 in 2007 (ECT Oekotoxikologie, 2007).

The study was performed using eight concentrations ranging from 0.156 to 20 mg/L, each tested in five replicates. Different exposure durations of 0, 24, 48, 72 and 96 h were tested (test system Desmodesmus subspicatus). The exposure duration of 72 h is considered for the key result. The cell concentration of each replicate was determined by measuring the absorption of the solutions with a spectral photometer. Since no calibration curve data were available, fluorescence data were equated with cell numbers. Yield, growth rate and integral of the biomass were determined from the cell densities at the respective observation times.

Analytical monitoring:

No chemical analysis of the test item concentrations was performed. However, based on the very high water solubility of the compound (miscible in a 1 : 1 ratio mixture (w/w) with water (at 20°C)), the very low adsorption potential (read across with n-butylamine (source): experimentally determined Koc from batch-equilibrium test: approx. 43.2 L/kg) and being not volatile (fully ionized at environmental pH based on a pKa of 10.8) it may be assumed with high confidence that the test item was stable during the test.

The following results were determined in this study:

EC50 (yield; 72 h): 4.13 (95% CI: 3.78-4.51) mg/L

EC10 (yield; 72 h) 0.887 (95% CI: 0.725-1.05) mg/L

NOEC (yield, 72 h): 1.25 mg/L

EC50 (growth rate; 72 h): 18.9 (18.2-19.6) mg/L

EC10 (growth rate; 72 h): 2.56 (2.41-2.70) mg/L

NOEC (growth rate, 72 h): 1.25 mg/L

EC50 (biomass integral; 72 h): 5.84 (4.94-6.97) mg/L

EC10 (biomass integral; 72 h): 1.13 (0.739-1.52) mg/L

NOEC (biomass integral): 0.625 mg/L

Description of key information

Toxcity of isopropylamine towards freshwater green algae (German industrial standard test guideline DIN 38412, part 9 (BASF 1988); GLP; re-evaluated according to OECD 201 in 2007 (ECT Oekotoxikologie, 2007)).

EC50 (growth rate; 72 h): 18.9 mg/L

EC10 (growth rate; 72 h): 2.56 mg/L

NOEC (growth rate, 72 h): 1.25 mg/L

No data is available for marine algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

18.9 mg/L

EC10 or NOEC for freshwater algae:

2.56 mg/L

Additional information

The substance isopropylamine was tested for algal toxicity in a reliable study performed compliant with GLP according to the German industrial standard test guideline DIN 38412, part 9 (BASF 1988).

The study was statistically re-evaluated according to OECD 201 in 2007 (ECT Oekotoxikologie, 2007).

The study was performed using eight concentrations ranging from 0.156 to 20 mg/L, each tested in five replicates. Different exposure durations of 0, 24, 48, 72 and 96 h were tested (test system Desmodesmus subspicatus). The exposure duration of 72 h is considered for the key result. The cell concentration of each replicate was determined by measuring the absorption of the solutions with a spectral photometer. Since no calibration curve data were available, fluorescence data were equated with cell numbers. Yield, growth rate and integral of the biomass were determined from the cell densities at the respective observation times.

Analytical monitoring:

No chemical analysis of the test item concentrations was performed. However, based on the very high water solubility of the compound (miscible in a 1 : 1 ratio mixture (w/w) with water (at 20°C)), the very low adsorption potential (read across with n-butylamine (source): experimentally determined Koc from batch-equilibrium test: approx. 43.2 L/kg) and being not volatile (fully ionized at environmental pH based on a pKa of 10.8) it may be assumed with high confidence that the test item was stable during the test.

The following results were determined in this study:

EC50 (yield; 72 h): 4.13 (95% CI: 3.78-4.51) mg/L

EC10 (yield; 72 h) 0.887 (95% CI: 0.725-1.05) mg/L

NOEC (yield, 72 h): 1.25 mg/L

EC50 (growth rate; 72 h): 18.9 (18.2-19.6) mg/L

EC10 (growth rate; 72 h): 2.56 (2.41-2.70) mg/L

NOEC (growth rate, 72 h): 1.25 mg/L

EC50 (biomass integral; 72 h): 5.84 (4.94-6.97) mg/L

EC10 (biomass integral; 72 h): 1.13 (0.739-1.52) mg/L

NOEC (biomass integral): 0.625 mg/L