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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental phase: 2019-09-13 to 2020-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008, amended by Commission Regulation (EU) 2017/735 of 14 February 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 7, 14 and 20 and of the expired test preparations on Days 1, 8, 15 and 21. All samples were stabilized by the addition of 0.01 M ammonium formate and 0.1% formic acid solution and stored frozen prior to analysis.
Duplicate samples were taken and stored frozen should any further analysis be required.
Vehicle:
no
Details on test solutions:
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give test solutions of 0.10, 0.32, 1.0, 3.2 and 10 mg/L. The pH of the 100 mg/L stock solution was checked and adjusted to 7.9 ± 0.3 prior to the dilutions being performed. The pH of all subsequent dilutions was recorded.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M4 medium (according to OECD TG 211, Annex 2) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
no
Hardness:
Water hardness measured as CaCO3 was determined in the control and the highest treatment group (10 mg/L) in fresh and old media with the following results:
Control: 244-272 mg/L
MPMD (10 mg/L): 252-270 mg/L
Test temperature:
The temperature was maintained at approximately 20 °C to 22 °C throughout the test
pH:
Control group: pH 7.8-8.5
Treatment groups (over all treatment concentrations and media preparations): pH 7.7-8.6
Dissolved oxygen:
= 8.5 mg O2/L throughout the study
Salinity:
n.a.
Conductivity:
not reported
Nominal and measured concentrations:
Nominal concentrations [mg/L]: 0.1, 0.32, 1.0, 3.2, 10.
Geometric mean measured concentrations [mg/L]: 0.078, 0.28, 0.89, 3.0, 9.3.
Details on test conditions:
See section "Any other information on materials and methods incl. tables".
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: extrapolated from concentration-response curve, 95% CI: 1.6 – 76 mg/L
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: only inadvertent parental immobilization observed in the control (n=1) and some of the treatment concentrations (no dose response relationship)
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction & parental length
Duration:
21 d
Dose descriptor:
other: EC5
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI: 0.38 – 3.5 mg/L
Duration:
21 d
Dose descriptor:
other: EC8
Effect conc.:
5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI: 1.1 – 24 mg/L
Details on results:
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. On Day 10 in the 0.10 mg/L test group young produced by one adult were observed to be small and pale, and on Day 17 in the 10 mg/L test group young produced by one adult were observed to be small. Given that these were transient effects that were not concentration dependent, it was considered that they were due to natural causes.
Young were first produced in the control test group on Day 8 of the test.
There were no unhatched eggs or dead young in any of the test groups.
For further information, see section "Any other information on results incl. tables"
Results with reference substance (positive control):
n.a.
Reported statistics and error estimates:
Analysis of Parental Mortality
Results from the control and each test group were compared using Qualitative Trend Analysis by Contrasts and the Fishers Exact Binomial Test with Bonferroni Correction. The analysis of contrasts did not reveal a linear trend.
No significant differences (P>=0.05) were found between the control and any of the test groups in terms of parental mortality. Therefore, the mortalities that were observed were classed as inadvertent for the purposes of the test.

Analysis of Numbers of Live Young Produced per Adult
Results from the control and each test group were compared using Shapiro Wilk’s test on Normal Distribution, Levene’s Test on Variance Homogeneity, Trend Analysis by contrasts and Williams Multiple Sequential t-test Procedure. The normality and variance homogeneity checks were passed and the analysis of contrasts revealed a linear trend. Accordingly, the Williams Multiple Sequential t-test Procedure was used to test for significance in relation to inhibition of reproduction by the test item compared to the control.
No significant differences (P>=0.05) were found between the control and any of test groups in terms of the number of live young produced per adult by Day 21 using the above methods of statistical analysis.

Analysis of the Daphnia Length Data
Results from the control and each test group were compared using Shapiro Wilk’s test on Normal Distribution, Levene’s Test on Variance Homogeneity, Trend Analysis by Contrasts and Dunnett’s Multiple t-test Procedure. The normality and variance homogeneity checks were passed, and the analysis of contrasts did not reveal a linear trend.
No statistically significant differences (P>=0.05) were found between the control and each test group in terms of length of the surviving parental daphnids on Day 21 of the test, using the above methods of statistical analyses.

Range-finding Test
No immobilization was observed in the control group, however, a single immobilized daphnia was observed in the 0.10 and 1.0 mg/L test groups and all daphnids were observed to be immobilized in the 10 and 100 mg/L test groups by the end of the test. Sub-lethal effects of exposure were observed at test concentrations of 1.0 and 10 mg/L. These responses were that parents were observed to be smaller or paler in coloration (see table 3 in the attached illustration).
Based on this information, test concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L were selected for the initial experiment.
Chemical analysis of the test preparations on Days 0 showed that near nominal concentrations were obtained. Analysis of the test preparations on Day 3 showed that concentrations ranged from 15% to 151% of nominal, suggesting that at the lower levels, the test item was unstable under test conditions.


 


Definitive Test
Based on the results of a preliminary range-finding test, test concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L were selected for the initial experiment. An inconsistent immobilization pattern was observed in this test, therefore the test was repeated using a concentration range of 0.10, 0.32, 1.0, 3.2 and 10 mg/L to ensure that any toxicity was appropriately captured.



Verification of Test Concentrations
Analysis of the fresh test preparations on Days 0, 7, 14 and 20 showed measured test concentrations to range from 0.081 to 11 mg/L. A decline in measured test concentration of some of the lower aged test preparations on Days 1, 8, 14 and 21 was observed with measured concentrations between 0.035 and 11 mg/L. Hence, it was considered appropriate to calculate the results based on the geometric mean measured test concentration. It was noted that the highest level tested (10 mg/L) remained near nominal throughout the duration of the test.
The following table reports the measured concentrations in detail:











































































































































































































































































































Sample
Description
Time point (days)MPMD Detected
(mg/L)
Percentage of Nominal Concentration Detected (%)
Control0 (fresh)ND-
0.10 mg/L 0.08181
0.32 mg/L 0.31598
1.0 mg/L 1.010101
3.2 mg/L 3.230101
10 mg/L 8.36084
Control1 (old)ND-
0.10 mg/L 0.03535
0.32 mg/L 0.18457
1.0 mg/L 0.74074
3.2 mg/L 2.63082
10 mg/L 10.200102
Control7 (fresh)ND-
0.10 mg/L 0.09696
0.32 mg/L 0.28689
1.0 mg/L 0.90691
3.2 mg/L 3.01094
10 mg/L 8.27083
Control8 (old)ND-
0.10 mg/L 0.05959
0.32 mg/L 0.29492
1.0 mg/L 0.84584
3.2 mg/L 3.07096
10 mg/L 8.21082
Control14 (fresh)ND-
0.10 mg/L 0.09999
0.32 mg/L 0.28990
1.0 mg/L 0.87087
3.2 mg/L 2.75086
10 mg/L 9.25093
Control15 (old)ND-
0.10 mg/L 0.07676
0.32 mg/L 0.26483
1.0 mg/L 0.83083
3.2 mg/L 3.06096
10 mg/L 7.98080
Control20 (fresh)ND-
0.10 mg/L 0.106106
0.32 mg/L 0.30495
1.0 mg/L 0.99399
3.2 mg/L 3.17099
10 mg/L 11.200112
Control21 (old)ND-
0.10 mg/L 0.08888
0.32 mg/L 0.27586
1.0 mg/L 0.93193
3.2 mg/L 2.97093
10 mg/L 10.800108

Accordingly, the geometric mean measured test concentrations were determined to be [mg/L]: 0.078, 0.28, 0.89, 3.0, 9.3.


 


Validity criteria
All validity criteria of OECD TG 211 were fulfilled:
Control mortality (<= 20%): 10%
Mean number of live young per surviving adult (control group; >=60 after 21 days): 169
Coefficient of variation for control group* (<=25%): 6%
No ephippia produced: true
Dissolved oxygen (>3 mg O2/L): ≥8.5 mg O2/L
pH (control group; 6 to 9, variation <=1.5): 7.8 to 8.5, 0.7


 


Lethal Effects on the Parental Generation (P1)
A single mortality was observed in the control group. Mortality was also observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However, statistical analysis of the mortality data using Qualitative Trend Analysis by Contrasts and the Fishers Exact Binomial Test with Bonferroni Correction showed that the observed mortalities were not significantly different (P>= 0.05) when compared to the control group and as such were classed as inadvertent mortalities with regard to the analysis. This method of analysis was chosen as the analysis of contrasts did not reveal a linear trend.
No mortalities occurred at the 0.32 and 3.2 mg/L test groups throughout the test.
Accordingly, the following ECx (immobilization) values based on the geometric mean measured test item concentrations were estimated by inspection of the data:
EC10 (parental immobilization; 21 days) > 9.3 mg/L
NOEC (parental immobilization; 21 days) >= 9.3 mg/L


 


Sub-lethal Effects on the Parental Generation (P1)
There was no significant effect on size and color of the daphnids in any of the concentrations employed in the test. A single daphnia in the 0.10 mg/L test group was observed to be pale on Day 21, and a single daphnia in the 10 mg/L test group was observed to be small on Days 3-8. Given that these were transient effects that were not concentration dependent, it was considered that they were due to natural causes.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P>= 0.05) between the control and the 0.078, 0.28, 0.89, 3.0 and 9.3 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item.
Body length data (day 21) for adults are given in the attached illustration (table 2).


 


Effects on Reproduction
Given that all parental mortality was determined to be accidental/inadvertent, the results are the same in terms of young produced per adult surviving to the end of the test versus young produced per adult which did not die accidentally/inadvertently.
Analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P>= 0.05) from each test group. However, the significance was just barely missed considering that the % MDD (minimal detectable difference) according to the Williams sequential t-test procedure was -7.6% for the highest tested concentration (9.3 mg/L) and % inhibition in relation to the control was 7.1%. Calculating inhibition in relation to the smoothed value (see below and / or methods section; 170.75) would result in 8.0% inhibition and thus significance at 9.3 mg/L (i.e. NOEC).


Prior to concentration-response modelling of the reproduction data, the control and 0.078 mg/L data was smoothed based on the arithmetic mean value of the "mean reproduction per surviving adult on day 21" for control and 0.078 mg/L according to US EPA (2002; 1994). Smoothing was performed to account for higher mean reproduction at 0.078 mg/L compared to control and to provide a stable baseline for dose response modelling. Details on the method are given in section "Any other information on materials and methods incl. tables".
Original and smoothed values for offspring per parental animal surviving till day 21 are as follows:


















































































































































 Treatment mg/LNumber of offspring per surviving adult day 21
ReplicateControl0.0780.280.8939.3
1186173170154144--
2167174174183181--
3166176173190155181
4173--157169155157
5177171181165153115
6--159165152140188
7158--162--177152
8178174146160178136
9152182150179171160
10164171196169169167
Cumulative number of offspring from adults surviving till day 21152113801674152116231256
No of surv. Adults98109108
Mean per surv. Ad.169172.5167.4169162.3157
Standard Deviation10.66.514.913.014.823.5

Smoothed values (control / 0.078 mg/L) used for dose-response modelling:


















































































































































 Treatment mg/LNumber of offspring per surviving adult day 21
ReplicateControl0.0780.280.8939.3
1187.93171.24170154144--
2168.73172.23174183181--
3167.72174.21173190155181
4174.79--157169155157
5178.83169.27181165153115
6--157.39165152140188
7159.64--162--177152
8179.84172.23146160178136
9153.57180.15150179171160
10165.70169.27196169169167
Cumulative number of offspring from adults surviving till day 211536.7513661674152116231256
No of surv. Adults98109108
Mean per surv. Ad.170.75170.75167.4169162.3157
Standard Deviation10.76.414.913.014.823.5

Concentration-response modelling
A 2-param. normal CDF (cumulative distribution function) F(x) =NormalCDF(b1-log10(x)/b2 + zOpt) was fitted to the normalized data. An iteratively re-weighted non-linear regression with Poisson weighting (1/Y) was performed.
Analysis of Variance and Test for Lack of Fit for the 2-param. Normal CDF:



  •  p(F) = probability that the variance explained by the regression is due to chance = 0.002 ==> Since p(F|Regression) <= 0.05, a significant amount of variance is explained by the regression model;

  •  p(F|Lack of fit) = 0.076 ==> Since p(F|Lack of Fit) > 0.05, there is no significant lack of fit.


Accordingly, the fit was accepted.
The confidence limits of the EC5 used as a parameter were computed by means of the standard error of parameter b0; confidence limits for the remaining ECx were estimated by Monte-Carlo simulation using the parameter errors obtained from the inverse Hessian matrix (5000 runs).


Further details on reproduction data and their evaluation are given in the attached illustration.



The following ECx (reproduction) values based on the geometric mean measured test concentrations were calculated/extrapolated via concentration-response modelling or estimated from inspection of the reproduction data at 21 days (in case of EC50):
EC5 (reproduction; 21 days) = 1.2 mg/L (95% CI: 0.38 – 3.5);
EC8 (reproduction; 21 days) = 5.0 mg/L (95% CI: 1.1 – 24);
EC10 (reproduction; 21 days) > 9.3 mg/L, extrapolated = 11 mg/L (95% CI: 1.6 – 76);
EC50 (reproduction; 21 days) > 9.3 mg/L;
NOEC (reproduction; 21 days) >= 9.3 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The following results were obtained for the test item MPMD (2-methylpentane-1,5-diamine) from a Daphnia magna reproduction test (OECD TG 211; GLP) based on geometric mean measured concentrations:

EC5 (reproduction; 21 days) = 1.2 mg/L (95% CI: 0.38 – 3.5);
EC8 (reproduction; 21 days) = 5.0 mg/L (95% CI: 1.1 – 24);
EC10 (reproduction; 21 days) > 9.3 mg/L, extrapolated = 11 mg/L (95% CI: 1.6 – 76);
EC50 (reproduction; 21 days) > 9.3 mg/L;
NOEC (reproduction, parental length, adult mortality; 21 days) >= 9.3 mg/L.
Executive summary:

A study on Daphnia magna reproduction with the test item MPMD (2-methylpentane-1,5-diamine) was performed compliant with GLP according to OECD TG 211 (2012) and EU method C.20 (2017) to assess the chronic toxicity of the test item to aquatic invertebrates.
Based on the results of a preliminary range-finding test and initial test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L for a period of 21 days in a semi-static test design (renewal daily). Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.9 with HCl before use in the test. Test item concentrations in fresh and used media were analytically verified on four instances evenly distributed over the total duration of the test.
The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and GEMMA Micro 300 fish food suspension.


Analysis of the fresh test preparations on Days 0, 7, 14 and 20 showed measured test concentrations to range from 0.081 to 11 mg/L. A decline in measured test concentration of some of the lower 'aged' test preparations on Days 1, 8, 14 and 21 was observed with overall measured concentrations between 0.035 and 11 mg/L. Hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration. It was noted that the highest level tested (10 mg/L) remained near nominal throughout the duration of the test. Accordingly, the geometric mean measured test concentrations were determined to be 0.078, 0.28, 0.89, 3.0, 9.3 mg/L.
All the validity criteria of the testing guideline were met.


Parental mortalities occurred in the control (n=1), 0.078 mg/L (n=2), 0.89 mg/L (n=1) and 9.3 mg/L (n=2). Based on statistical analysis confirming that all parental mortalities were accidental/inadvertent, the results are the same in terms of young produced per adult surviving to the end of the test versus young produced per adult which did not die accidentally/inadvertently.
Statistical analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P>= 0.05) from each test group. However, the significance level was just barely missed considering that the % MDD (minimal detectable difference) according to the Williams sequential t-test procedure was -7.6% for the highest tested concentration (9.3 mg/L) and % inhibition in relation to the control was 7.1%. Reproduction rate at the lowest test item concentration (0.078 mg/L) was higher than in the control, and calculating inhibition in relation to the arithmetic mean reproduction value of control and 0.078 mg/L would result in 8.0% inhibition and thus significance at 9.3 mg/L (i.e. NOEC). It is unclear why parental mortality was observed in the range finding test at the concentration of 10 mg/L. Data from the definitive test showed no such effect at this level and considering the increased replicates and associated increased statistical power, those final results are considered valid and reliable. However, because of the mortality results from the preliminary test, the highest test concentration was not optimal set (too low). Inhibition effects on reproduction were just starting to become obvious at the highest tested concentration. Therefore, low effect concentrations were derived using concentration-response modelling using ToxRat v.3.3.
Overall, exposure of Daphnia magna to MPMD gave the following results based on geometric mean measured test item concentrations:


EC5 (reproduction; 21 days) = 1.2 mg/L (95% CI: 0.38 – 3.5);
EC8 (reproduction; 21 days) = 5.0 mg/L (95% CI: 1.1 – 24);
EC10 (reproduction; 21 days) > 9.3 mg/L, extrapolated = 11 mg/L (95% CI: 1.6 – 76);
EC50 (reproduction; 21 days) > 9.3 mg/L;
NOEC (reproduction, parental length, adult mortality; 21 days) >= 9.3 mg/L.

Description of key information

The following results were obtained for the test item MPMD (2-Methylpentane-1,5-diamine) from a Daphnia magna reproduction test (OECD TG 211; GLP) based on geometric mean measured concentrations:


EC5 (reproduction; 21 days) = 1.2 mg/L (95% CI: 0.38 – 3.5);
EC8 (reproduction; 21 days) = 5.0 mg/L (95% CI: 1.1 – 24);
EC10 (reproduction; 21 days) > 9.3 mg/L, extrapolated = 11 mg/L (95% CI: 1.6 – 76);
EC50 (reproduction; 21 days) > 9.3 mg/L;
NOEC (reproduction, parental length, adult mortality; 21 days) >= 9.3 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
>= 9.3 mg/L

Additional information

A study on Daphnia magna reproduction with the test item MPMD (2-Methylpentane-1,5-diamine) was performed compliant with GLP according to OECD TG 211 (2012) and EU method C.20 (2017) to assess the chronic toxicity of the test item to aquatic invertebrates.
Based on the results of a preliminary range-finding test and initial test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L for a period of 21 days in a semi-static test design (renewal daily). Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.9 with HCl before use in the test. Test item concentrations in fresh and used media were analytically verified on four instances evenly distributed over the total duration of the test.
The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and GEMMA Micro 300 fish food suspension.


Analysis of the fresh test preparations on Days 0, 7, 14 and 20 showed measured test concentrations to range from 0.081 to 11 mg/L. A decline in measured test concentration of some of the lower 'aged' test preparations on Days 1, 8, 14 and 21 was observed with overall measured concentrations between 0.035 and 11 mg/L. Hence, it was considered appropriate to calculate the results based on the geometric mean measured test concentration. It was noted that the highest level tested (10 mg/L) remained near nominal throughout the duration of the test. Accordingly, the geometric mean measured test concentrations were determined to be 0.078, 0.28, 0.89, 3.0, 9.3 mg/L.
All the validity criteria of the testing guideline were met.


Parental mortalities occurred in the control (n=1), 0.078 mg/L (n=2), 0.89 mg/L (n=1) and 9.3 mg/L (n=2). Based on statistical analysis confirming that all parental mortalities were accidental/inadvertent, the results are the same in terms of young produced per adult surviving to the end of the test versus young produced per adult which did not die accidentally/inadvertently.
Statistical analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P>= 0.05) from each test group. However, the significance level was just barely missed considering that the % MDD (minimal detectable difference) according to the Williams sequential t-test procedure was -7.6% for the highest tested concentration (9.3 mg/L) and % inhibition in relation to the control was 7.1%. Reproduction rate at the lowest test item concentration (0.078 mg/L) was higher than in the control, and calculating inhibition in relation to the arithmetic mean reproduction value of control and 0.078 mg/L would result in 8.0% inhibition and thus significance at 9.3 mg/L (i.e. NOEC). It is unclear why parental mortality was observed in the range finding test at the concentration of 10 mg/L. Data from the definitive test showed no such effect at this level and considering the increased replicates and associated increased statistical power, those final results are considered valid and reliable. However, because of the mortality results from the preliminary test, the highest test concentration was not optimal set (too low). Inhibition effects on reproduction were just starting to become obvious at the highest tested concentration. Therefore, low effect concentrations were derived using concentration-response modelling using ToxRat v.3.3.
Overall, exposure of Daphnia magna to MPMD gave the following results based on geometric mean measured test item concentrations:


EC5 (reproduction; 21 days) = 1.2 mg/L (95% CI: 0.38 – 3.5);
EC8 (reproduction; 21 days) = 5.0 mg/L (95% CI: 1.1 – 24);
EC10 (reproduction; 21 days) > 9.3 mg/L, extrapolated = 11 mg/L (95% CI: 1.6 – 76);
EC50 (reproduction; 21 days) > 9.3 mg/L;
NOEC (reproduction, parental length, adult mortality; 21 days) >= 9.3 mg/L.