# Analysis of Chronic Toxicity Data **and Acute Chronic Ratios (ACRs) in Support of Deriving Chronic HC5s:**  **Acetylcholinesterase Inhibitors** 

David R Mount and Russell J Erickson Office of Research and Development, Duluth, MN

**1** 

# Presentation to the FIFRA Scientific Advisory Panel January 31, 2012

# Acute Chronic Ratios (ACRs)

• Ratio of acute effect concentration to chronic effect concentration

• Acute effect conc / chronic (threshold) effect conc



# • Acute effect concentration typically the 96

- h LC50 (48

- h for some species)

• Chronic effect concentration may be

• Chronic NOEC

• Geomean of chronic NOEC and LOEC

• Chronic effect point estimate such as EC20



# • Will use subscripts to differentiate

• ACR NOEC 

# , ACR GM 

# , ACR EC20 

# , respectively **2**

# Distributions of Toxicity Data**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



### Ac **ute**



## 3



## Effect Concentration (  **g/L) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 0.03** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50**  **70**



## Acute HC5

# Distributions of Toxicity Data**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



### Chronic

**Ac ute** 



## 4 

## Effect Concentration (  **g/L) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 0.03** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50**  **70**



## Acute HC5 Chronic HC5

# Distributions of Toxicity Data**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



### Chronic

**Ac ute** 



## 5 

## Effect Concentration (  **g/L) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 0.03** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50**  **70**



## Acute HC5 Chronic HC5

ACR HC5

# Distributions of Toxicity Data**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



### Chronic

**Ac ute** 



## ACR

Large

**6** 

## Effect Concentration (  **g/L) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 0.03** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50**  **70**



## Acute HC5 Chronic HC5

ACR HC5

# Distributions of Toxicity Data**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



### Chronic

**Ac ute** 



## ACR

Large

**7** 

## Effect Concentration (  **g/L) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 0.03** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50**  **70**



## Acute HC5 Chronic HC5

ACR HC5



## ACR Small

# Distributions of Toxicity Data**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



### Chronic

**Ac ute** 



## ACR

Large 

# ACRs vary widely within and across chemicals; how do we estimate ACR HC5 

# when chronic data are limited or unavailable?

**8** 

## Effect Concentration (  **g/L) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 0.03** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50**  **70**



## Acute HC5 Chronic HC5

ACR HC5



## ACR Small

# Scope of Current ACR Analysis

• Presume that ACRs reflect underlying toxicokinetic and toxicodynamic characteristics of chemicals

• Hypothesize considering mode of action (MOA) can help reduce, or at least parse, uncertainty in estimation of ACR HC5



# • Begin by focusing on chronic toxicity data for AChE inhibiting chemicals

**9**

# Data **Compilation**

• 119 chronic tests for AChE inhibitors compiled from:

• Existing ORD data compilations

• FIFRA DERs

• ECOTOX searches



# • Treatment level data for each endpoint reviewed and screened (details in Appendix)

• 73 tests passed screening and had defined NOEC and LOEC values

• NOEC/LOEC for individual tests based on most sensitive endpoint relating to survival, growth, or reproduction

**10**

# Data **Distribution**

Americamysis bahia Ceriodaphnia

dubia Daphnia

magna Cyprinodon

variegatus Jordanella

floridae Menidia

beryllina Menidia

peninsulae Oncorhynchus

mykiss Opsanus

beta Pimephales

promelas
Aminocarb 

### 1
Carbaryl (Sevin) 

### 1

Carbofur an 

### 2 1

Methom yl 

### 1 1 1 1

Pr opo xur (Baygon) 

### 1
Azinphos

- meth yl 

### 1
Carbophenothion 

### 1
Chlorpyrif os (Dur sban) 

### 2 1 6 1 1 1 6

Diazinon 

### 1 1 1 3



## 11 Diazinon 

### 1 1 1 3
Dichlorvos (DDVP) 

### 1 1 1

Disulfoton 

### 1 1 1 2
Ethopr op 

### 1 1 1

Fensulfothion 

### 1

Fenthion 

### 1

Fonofos 

### 1 1 1 1

Malathion 

### 1 1 1 1

Methidathion 

### 1 2
Methyl par athion 

### 1 2 1

Naled 

### 1 1 1

Parathion 

### 1

Phosmet 

### 1 1

Pr ofenofos 

### 1 1

Te rbufos 

### 1

# Acute Data

• Acute value for ACR selected based on following priorities:

• 1) value from concurrent study

• 2) value from same laboratory but from a different time

• 3) value (or geomean ) from ICE database, with preference for

• Meets ICE minimum modeling requirements

• Measured chemistry

• Flow

- through methods

• Following EPA/OPP methods

**12**

# Aggregate ACR Distribution**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 70 **80 90**  **95 98**  **99 99.8** 



## 13



## Ac **ute Chronic Ratio (LC50/GM) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 10000** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 0.2 **0.5 1**  **2 5**  **10 20**  **30 50** 

# Aggregate ACR Distribution**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 70 **80 90**  **95 98**  **99 99.8** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 50 **70 80**  **90 95**  **98 99**  **99.8**



## 14



## Ac **ute Chronic Ratio (LC50/GM) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 10000** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 0.2 **0.5 1**  **2 5**  **10 20**  **30 50** 



## Ac **ute Chronic Ratio (LC50/GM) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 10000** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 0.2 **0.5 1**  **2 5**  **10 20**  **30 50** 



### Inv **ertebrates Fish** 

# Fish ACRs > Invertebrate ACRs**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



## 15



## Ac **ute Chronic Ratio (LC50/GM) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 10000** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50** 

**Invertebrates**
**Fish**

# OP ACRs ≈ Carbamate **ACRs C** **u m** **u l** **a t** **i v** **e P** **e r** **c e** **n t** 

**70 80**  **90 95**  **98 99**  **99.8**



## 16



## Ac **ute Chronic Ratio (LC50/GM) 0.1**  **1 10**  **100 1000**  **10000 C** **u m** **u l** **a t** **i v** **e P** **e r** **c e** **n t** 

**0.5 1**  **2 5**  **10 20**  **30 50** 

**Invert Carbamate**
**Invert OP**
**Fish Carbamate**
**Fish OP**

# Acute **Sensitivity to AChE Inhibitors**

**Planktonic Inv**  **ertebrates (**  **Daphnia)**

**Fish**Frequency 0.8 1.0 1.2 

## Fish roughly 100x less **acutely sensitiv e**  **than daphnids**  **to AC**  **hE inhibitors** 

**17** 

## Relative Acute LC50 for AChE Inhibitors 1 10 100 1000 10000Frequency

0.0 0.2 0.4 0.6



## Extracted from **Erickson (Appendix D)** 

# Fish GMs > D. magna GMs**F i** **s h** **G M** **( u** **g /** **L )** 



## 10

**100 1000** 

**Fish chronic less sensitiv**  **e**

**D. magna GM = Fish GM** 



## 18 

# D. magna **GM (**  

# g/L) **0.01 0.1**  **1 10**  **100 1000** **F i** **s h** **G M** **( u** **g /** **L )** 



## 0.01 **0.1 1** 

**Fish chronic more sensitiv**  **e D. magna**  **GM = Fish GM**

# Large Fish ACRs ≠ High Sensitivity**N O** **E C** **f r** **o m** **F i** **s h** **C h** **r o** **n i** **c T** **e s** **t (** **g /** **L )** 



### 100

**1000 Fish NOEC** 
**Fish Unbounded NOEC**

**Fish chronic less sensitive**  

# 1:1 **Line**

**19** 

## Daphnia magna **48-h LC50 (** 

#  **g/L) 0.1**  **1 10**  **100 N** **O E** **C f** **r o** **m F** **i s** **h C** **h r** **o n** **i c** **T e** **s t** **(**



### 0.1 **1 10** 

**Fish chronic more sensitive** 

# Large Fish ACRs ≠ High Sensitivity**N O** **E C** **f r** **o m** **F i** **s h** **C h** **r o** **n i** **c T** **e s** **t (** **g /** **L )** 



### 100

**1000 Fish NOEC** 
**Fish Unbounded NOEC**

**Fish chronic less sensitive**  

## Estimated **Ac ute**  **HC5 (Mean EF)** 

**20** 

## Daphnia magna **48-h LC50 (** 

#  **g/L) 0.1**  **1 10**  **100 N** **O E** **C f** **r o** **m F** **i s** **h C** **h r** **o n** **i c** **T e** **s t** **(**



### 0.1 **1 10** 

**Fish chronic more sensitive**  

## (Mean EF)

# Large Fish ACRs ≠ High Sensitivity**N O** **E C** **f r** **o m** **F i** **s h** **C h** **r o** **n i** **c T** **e s** **t (** **g /** **L )** 



### 100

**1000 Fish NOEC** 
**Fish Unbounded NOEC**

**Fish chronic less sensitive** 



## Estimated

**21** 

## Daphnia magna **48-h LC50 (** 

#  **g/L) 0.1**  **1 10**  **100 N** **O E** **C f** **r o** **m F** **i s** **h C** **h r** **o n** **i c** **T e** **s t** **(**



### 0.1 **1 10** 

**Fish chronic more sensitive**  

## Estimated

**Ac ute**  **HC5 (90**  **th** 

## %tile)

# Saltwater and Freshwater ACRs**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**70 80**  **90 95**  **98 99**  **99.8**



## 22



## Ac **ute-Chronic Ratio (LC50/GM) 0.1**  **0.3 1**  **3 10**  **30 100**  **300 1000**  **3000 10000** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**

**0.5 1**  **2 5**  **10 20**  **30 50** 

**SW Invertebrates**
**FW Invertebrates**
**SW Fish**
**FW Fish**

# Invert ACRs by Species**C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 50

**70 80**  **90 95**  **98**



## 23



## Ac **ute Chronic Ratio (LC50/EC20) 0.1**  **0.2 0.5**  **1 2**  **5 10**  **20 50**  **100 200**  **500 1000** **C u** **m u** **l a** **t i** **v e** **P e** **r c** **e n** **t**



### 2 **5 10**  **20 30**  **50**

**Americamysis bahia Daphnia magna**  **Ceriodaphnia dubia**

# Paired Comparison of Mysid/magna

Chemical Mysid ACR D. magna ACR Mysid/magna Log Mysid/magna Chlorpyrif os (Dur sban) 1.75 3.84 0.456

- 0.341

Diazinon 2.64 4.97 0.532

- 0.274 Ethoprop 6.13 1.56 3.941 0.596
Fonof os 7.33 2.75 2.664 0.426
Methidathion 26.71 9.19 2.907 0.463
Methom yl 8.84 3.67 2.411 0.382

**24** 

## Methom yl 8.84 3.67 2.411 0.382
Naled 6.73 2.38 2.832 0.452
Prof enof os 8.42 6.45 1.306 0.116

mean = 0.228

SD = 0.357

2

- ta iled t p=0.05 df=7 2.365 t = 1.803

Not significantly diff er ent under 2

- ta iled te st at p=0.05

# Selecting an Invertebrate ACR

• Uncertain whether mysid ACRs are larger than daphnid ACRs

• Even if they are, is this a FW v SW difference or a species difference?

• Is it likely that there are other FW invertebrates that would have mysid

- like ACRs?



# • Under 1985 guidelines, final ACR for AChE inhibitor would average daphnid and mysid ACRs

• Seems reasonable to select AChE inhibitor ACR HC5 

# from combined invert distribution **25**

# Invertebrate **ACRs by Endpoint C** **u m** **u l** **a t** **i v** **e P** **e r** **c e** **n t** 



### 70

**80 90**  **95 98**  **99**



# AC **R NOEC** 



# AC **R GM**  

# AC **R EC20** 



## 26



# AC **R 0.1**  **0.2 0.5**  **1 2**  **5 10**  **20 50**  **100 200**  **500 C** **u m** **u l** **a t** **i v** **e P** **e r** **c e** **n t** 



### 1 **2 5**  **10 20**  **30 50** 

# Comparison of ACRs

**AC R**  **Distribution Source/T**  **ype Percentile** 

**50th 80th**  **90th**
Present Analysis: AChE inhibitors, all species ACR GM 

## 13.9 61.1 124 AChE inhibitors, fish only ACR GM 

## 29.2 79.0 247 AChE inhibitors, invertebrates only ACR NOEC 

## 7.98 17.9 30.6 AChE inhibitors, invertebrates only ACR GM 

## 5.91 13.6 18.8 AChE inhibitors, invertebrates only ACR EC20 

## 3.88 8.63 18.0

**27**

# Comparison **to “Generalized” ACRs** 

• Raimondo et al. (2007)

• Evaluated distributions of ACRs in light of several variables, including MOA

• Comparisons were univariate



# • Host et al (1995)

• Assessed distribution of final ACRs (ACR HC5 

# ) from WQC documents (80 th 

# percentile used for GLI)



# • TenBrook et al. (2009)

• Subset of final ACRs selecting only chemicals used as pesticides (multiple MOA)

**28**

# Comparison of ACRs

**AC R**  **Distribution Source/T**  **ype Percentile** 

**50th 80th**  **90th**
Present Analysis: AChE inhibitors, all species ACR GM 

## 13.9 61.1 124 AChE inhibitors, fish only ACR GM 

## 29.2 79.0 247 AChE inhibitors, invertebrates only ACR NOEC 

## 7.98 17.9 30.6 AChE inhibitors, invertebrates only ACR GM 

## 5.91 13.6 18.8 AChE inhibitors, invertebrates only ACR EC20 

## 3.88 8.63 18.0

**29** Raimondo et al. (2007): Combined chemicals, all species ACR GM 

## 8.3 NR 79.5 AChE inhibitors, all species ACR GM 

## 6.4 NR 60.2

# Comparison of ACRs

**AC R**  **Distribution Source/T**  **ype Percentile** 

**50th 80th**  **90th**
Present Analysis: AChE inhibitors, all species ACR GM 

## 13.9 61.1 124 AChE inhibitors, fish only ACR GM 

## 29.2 79.0 247 AChE inhibitors, invertebrates only ACR NOEC 

## 7.98 17.9 30.6 AChE inhibitors, invertebrates only ACR GM 

## 5.91 13.6 18.8 AChE inhibitors, invertebrates only ACR EC20 

## 3.88 8.63 18.0

**30** Raimondo et al. (2007): Combined chemicals, all species ACR GM 

## 8.3 NR 79.5 AChE inhibitors, all species ACR GM 

## 6.4 NR 60.2

# Comparison of ACRs

**AC R**  **Distribution Source/T**  **ype Percentile** 

**50th 80th**  **90th**
Present Analysis: AChE inhibitors, all species ACR GM 

## 13.9 61.1 124 AChE inhibitors, fish only ACR GM 

## 29.2 79.0 247 AChE inhibitors, invertebrates only ACR NOEC 

## 7.98 17.9 30.6 AChE inhibitors, invertebrates only ACR GM 

## 5.91 13.6 18.8 AChE inhibitors, invertebrates only ACR EC20 

## 3.88 8.63 18.0

**31** Raimondo et al. (2007): Combined chemicals, all species ACR GM 

## 8.3 NR 79.5 AChE inhibitors, all species ACR GM 

## 6.4 NR 60.2
Host et al. (1995): Combined chemicals, freshwater FA CR 7.1 17.9 29.1 Combined chemicals, saltwater F ACR 4.4 13.5 24.2
Te nBrook et al. (2010) Combined chemicals, freshwater FA CR NR 12.4 NR

# Sources of Variability in ACRs

• ACR distributions contain variability from multiple sources:

• True differences among species

• True differences among chemicals

• Random variation in LC50 and chronic value (variation multiplicative in ratio)

**32 T** **o t** **a l** **R e** **p r** **o d** **u c** **t i** **o n** **a s** **P e** **r c** **e n** **t o** **f C** **o n** **t r** **o l** 



### 80

**100 120**  

# Survival Normalized Reproduction

Daphnia magna tests measuring total young or young/female

**Chronic LC50** 

**Exposure Relativ e to Chronic LC50**  **0.003 0.01**  **0.03 0.1**  **0.3 1**  **3 10** **T o** **t a** **l R** **e p** **r o** **d u** **c t** **i o** **n a** **s P** **e r** **c e** **n t** **o f** **C o** **n t** **r o** **l**



### 0 **20 40**  **60**

**Test 1**

**Test 2**
**Test 3**
**Test 4**
**Test 5**
**Test 6** 

## EC50 = 1.02

**EC20 = 0.77 EC0 = 0.48** 

**33 Y** **o u** **n g** **P e** **r R** **e p** **r o** **D a** **y a** **s P** **e r** **c e** **n t** **o f** **C o** **n t** **r o** **l**



### 80

**100 120**  

# Survival Normalized Reproduction

Daphnia magna tests measuring young per reproductive day

**Chronic LC50** 

**Exposure Relativ e to Chronic LC50**  **0.003 0.01**  **0.03 0.1**  **0.3 1**  **3 10** **Y o** **u n** **g P** **e r** **R e** **p r** **o D** **a y** **a s** **P e** **r c** **e n** **t o** **f C** **o n** **t r** **o l** 



### 0 **20 40**  **60**

Te st 1

Te st 2 Te st 3 Te st 4 Te st 5 Te st 6 Te st 7 

## EC50 = 1.24

**EC20 = 0.73 EC0 = 0.29** 

**34**

# Effect of Random Variation

• Monte Carlo simulation **AC R**  **Distribution Source/T**  **ype Percentile** 

**50th 80th**  **90th**
Present Analysis: AChE inhibitors, invertebrates only ACR GM 

## 5.91 13.6 18.8



# • Mean acute value = 5.91, mean chronic value = 1

• Appropriate variability uncertain; for example purposes, used “factor of 2” rule of thumb

• Log

- normal distributions with mean ± 2 SD equal to 0.5x and 2x of true value

**35**

# Monte Carlo Results

**AC R**  **Distribution Source/T**  **ype Percentile** 

**50th 80th**  **90th**
Present Analysis: AChE inhibitors, invertebrates only ACR GM 

## 5.91 13.6 18.8
Monte Carlo Simulation, true mean 5.91 Log

- normal

, mean ± 2 SD = 0.5x to 2x 5.91 11 .0 15.6

**36** 

# • Observed variability in ACRs for AChE inhibitors comparable to variability expected if assumed 2x variability is realistic

• May affect risk management decisions regarding percentile selection if upper tails of distribution result primarily from random variability rather than true differences in response

# Conclusions

• For AChE inhibitors, ACR distribution for fish higher than for invertebrates

• Despite this, an acute HC5 protective of invertebrates would also protect fish from chronic effects

• Acute HC5 and chronic HC5 driven by invertebrate data, so ACR HC5 

# should be selected from invertebrate ACRs

• For AChE inhibitors, MOA

- specific ACRs up to 2

- fold lower than generalized ACRs

**37**

# Questions?

**38**